WO2001025779A2

WO2001025779A2 - Electrophoresis device for analysing labelled molecules, in particular, biological molecules

Info

Publication number: WO2001025779A2
Application number: PCT/EP2000/009777
Authority: WO
Inventors: Wilhelm Ansorge; Josef Stegemann; Robert Ventzki; Christian Schwager
Original assignee: Europäisches Laboratorium für Molekularbiologie (EMBL)
Priority date: 1999-10-07
Filing date: 2000-10-05
Publication date: 2001-04-12
Also published as: WO2001025779A3; AU1021901A; DE19948391A1

Abstract

The invention relates to an electrophoresis device (10) for analysing molecules labelled with fluorescent markers, in particular, biological molecules. According to one aspect of the invention, said device is suitable for resolving at least four different fluorescent markers independently of one another using a detector field assigned to each marker, according to the respective gel electrophoresis lane and the respective fluorescent marker. To this end, spectral filters are balanced with one another with regard to their spectral selectivity, the fluorescent markers are balanced with one another with regard to their spectral absorption and emission selectivity and the wavelengths of the laser beams (60, 62, 64, 66) which are used to excite the fluorescent markers are balanced with one another in a corresponding manner.

Description

Electrophoresis device for analyzing labeled molecules, especially biological molecules

description

The present invention relates to an electrophoresis device for analyzing fluorescence marker-labeled molecules, in particular biological molecules (eg DNA, DNA fragments or proteins), which comprise a plurality of gel electrophoresis cells which run essentially parallel to one another and along a direction of molecular expansion. Webs, an energy source for applying an electrical potential at an initial region and an end region of the gel electrophoresis webs and a laser arrangement for exciting the fluorescence markers associated with the molecules, the laser arrangement in the electrophoresis mode generating laser radiation which, as at least one, essentially transversely to the The laser beam extending in the direction of the molecule cuts at least several of the gel electrophoresis tracks in order to excite fluorescence markers of the molecules present in a detection area of the respective gel electrophoresis track, as well as a detection arrangement for Detection of emission radiation emanating from the excited fluorescence markers, the detection arrangement having at least one detector field which extends essentially parallel to the at least one laser beam and which detects emission radiation guided to the detector field via a spectral filter and radiation guide arrangement. The detector field, which extends along a preferably linear detection path and enables spatially resolved detection of the emission radiation along the detection path defined by the course of the laser beam section cutting the gel electrophoresis paths (and therefore also referred to below as a linear detector field with regard to a linear detection path) , can be used by a number of detection elements, such as photodiodes or CCD Elements, or a matrix of several parallel rows of detection elements (such as photodiodes or CCD elements) can be formed. In principle, however, there are no restrictions with regard to the mode of operation and the functional principle of the detector field that enables the spatially resolved detection.

Such an electrophoresis device is known in several embodiments from WO96 / 2321 3, which gives a good overview of the background to the analysis of biological molecules by means of gel electrophoresis, as well as the basic functions and design options of such electrophoresis devices and the disclosure thereof is fully incorporated by reference into the disclosure of the present description of the invention. WO96 / 2321 3 discloses several electrophoresis devices which have two or three laser beam sources, apparently in order to be able to analyze two or three fluorescent dyes (fluorescent markers). In two embodiments, two laser beams from two separate laser light sources, which are essentially transverse to the direction of molecular expansion and essentially parallel to one another and are mutually offset in the direction of molecular expansion, are coupled into the electrophoresis gel, each of which is assigned a separate linear detector field and a separate spectral filter. Alternatively, linear CCD element fields and linear photodiode fields are provided as linear detector fields. WO96 / 2321 3 proposes the use of gradient index lens fields (specifically so-called SELFOC gradient-index microlens arrays) to collect and focus the emission light.

The simultaneous determination of two nucleic acid sequences using two differently labeled primer molecules for a sequencing reaction in a single reaction vessel was described by Wiemann et al. (Anal, biochem. 224 (1 995), 1 1 7-1 21). Other methods in which sequencing reactions using two Different fluorescent labels in a single reaction vessel, combined with a joint separation of these two different fluorescent labels, are described in DE 1 95 1 5 552 A1 and WO96 / 341 14. Detection systems containing two different lasers are used to evaluate the sequencing reactions. WO96 / 2321 3 obviously provides a detection system which enables an evaluation of such sequencing reactions.

For the technical background of DNA sequence analysis based on laser-assisted detection of fluorescent dye-labeled DNA fragments, reference can also be made to the following basic articles: Nature, Vol. 321, 1 2.06.1 986, pages 674-679 , LM Smith et al: "Fluorescence detection in automated DNA sequence analysis" and Journal of Biochemical and Biophysical Methods, Vol. 1 3, 1 986, pages 31 5-323, W. Ansorge et al: "A non-radioactive automated method for DNA sequence determination ". Further writings and articles important for understanding the technical background are mentioned in WO96 / 2321 3.

EP 0 275 440 B1 should also be mentioned, which deals with the minimization of the background fluorescence from the gel by appropriate selection of the fluorescent dye used and the laser wavelength used for fluorescence excitation.

In addition to a primer-specific fluorescent dye label, a base-specific fluorescent dye label is also used in practice, for which preferably fluorescent dye-labeled stop nucleotides (labeled chain termination molecules, in particular dideoxyribonucleoside triphosphates (dd NTPs)) or labeled deoxyribonucleoside triphosphates are used , Specifically for sequence determination based on a base specific Fluorescent dye labeling is offered by Perkin Elmer Biosystems (formerly Applied Biosystems) layer gel sequencing devices that excite the four different fluorescent dyes assigned to a clone for the four different nucleotides by means of a laser common to all dyes, and the resulting fluorescent radiation via four spectral filter arrangements each assigned to one dye to capture. However, due to strong spectral crosstalk, the four dyes cannot be detected independently of one another in a dye-resolved manner. A determination of the sequence is only possible on the basis of a complex software correction of the raw measurement data not yet resolving the dyes, which is based on the fact that the four specific, differently marked sequencing approaches of a clone are separated and analyzed together in a gel trace. A sample-specific detection, in which, for example, four different clones assigned and differently marked approaches are separated and analyzed in a gel trace, is not possible despite the software correction.

A major disadvantage of the base-specific labeling is moreover that the coupled dyes influence the mobility of the DNA fragments due to different chemical parameters and cause so-called "mobility shifts" which have to be corrected by software. The correction of the mobility shifts and the correction of the spectral crosstalk described above lead to errors and inaccuracies with the consequence of shorter reading lengths and a higher error rate. This means that sequencing must be carried out with higher redundancy. This means that the same sequence must be read several times.

The problem with the joint analysis of sequencing products labeled with different fluorescent dyes, generally of molecules labeled with different fluorescent dyes, in a single lane or gel track is generally that fluorescent dyes can be excited over a wide wavelength range and the excitation energy also return over a wide range. In addition, the excitation and emission spectra are subject to external conditions, which are given by parameters of the chemical environment of the dye, including the pH value, the temperature and the dielectric constant of the solution. It was therefore necessary to assume that a simultaneous quantitative recording of four or more independent dye spectra, as required, for example, for an exact evaluation of sequencing reactions, cannot be carried out.

Surprisingly, it has now been found that a skilful selection of the spectral filters used with regard to their spectral selectivity, the fluorescent markers with regard to their spectral absorption and emission selectivity and the wavelengths of the laser beams enables four or more fluorescent marker spectra (dye spectra) to be detected and evaluated differently from one another, so that, for example, a joint evaluation of four or more sequencing reactions in which different fluorescent dyes were used in each case is possible, specifically a joint evaluation of four or more sample-specific (in particular primer-specific) marked sequencing reactions. This is of great practical relevance, as the cost of carrying out the sequencing reactions can be significantly reduced by using four or more submersible markings. Both chemical and labor costs are reduced. This is particularly important in large-scale sequencing projects, such as the "Human Genome" project or other genome projects.

Accordingly, according to a first aspect, the invention provides an electrophoresis device for analyzing fluorescent markers. labeled molecules, in particular biological molecules, which comprises: a plurality of gel electrophoresis tracks substantially parallel to one another and along a molecular propagation direction, a source of energy for applying an electrical potential to a starting area and an end area of the gel electrophoresis tracks, a laser arrangement for exciting a plurality of different fluorescent markers assigned to the molecules, at least some of which have absorption wavelength ranges which are different from one another and / or emission wavelength ranges which differ from one another, the laser arrangement in electrophoresis mode generating laser radiation which is essentially transverse to the direction of molecular propagation and in parallel with one another and laser beams offset in the direction of molecular propagation, it cuts at least several of the gel electrophoresis tracks in order to detect the respective area in a detection area to stimulate fluorescent markers of the molecules present there associated with gel-electrophoresis path, a detection arrangement for gel-electrophoresis-path-resolved and fluorescence-marker-resolved detection of emission radiation emitted by the excited fluorescence markers, the detection arrangement displacing and preferably displacing a plurality of one another in the direction of molecular propagation has detector fields which extend essentially parallel to the laser beams and which are each assigned to a specific one of the laser beams and are designed for this purpose, along a detection path associated with the respective laser beam, preferably essentially parallel to this, at least a portion of those in the detection areas due to the excitation by the respective one Detect the laser beam that arises in a spatially resolved manner via an assigned spectral filter and radiation guide arrangement for the detector field. the spectral filter arrangement with regard to its spectral selectivity, the fluorescent markers with regard to their spectral absorption and emission selectivity, and the wavelengths of the laser beams in this way are coordinated with one another such that, using at least four laser beams and at least four detector fields, at least four different fluorescence markers can be detected independently of one another by means of an assigned detector field according to the respective gel electrophoresis path and the respective fluorescence marker.

In generalizing the concept of the invention on which the proposed electrophoresis device is based, an electrophoresis device for analyzing fluorescent marker-labeled molecules, in particular biological molecules, is also provided, which comprises: a plurality of gel electrophoresis cells which run essentially parallel to one another and along a direction of molecular propagation. Webs, an energy source for applying an electrical potential to a start region and an end region of the gel electrophoresis webs, a laser arrangement for exciting a plurality of different fluorescence markers assigned to the molecules, at least some of which have absorption wavelength ranges that are different from one another and / or different from one another Have emission wavelength ranges, the laser arrangement in the electrophoresis mode generating laser radiation, which is essentially transverse to the direction of molecular propagation of the laser beam len cuts at least several of the gel electrophoresis tracks in order to excite fluorescence markers of the molecules present in a detection area of the respective gel electrophoresis track, at least some of the laser beams being spatially offset from one another and / or occurring in offset time windows or / and wherein at least one laser beam is modulated to enable phase-sensitive detection or / and wherein at least some of the laser beams have different wavelengths from one another, a detection arrangement for gel electrophoresis path-resolved and fluorescence marker-resolved detection of radiation emitted by the excited fluorescence markers, the detection arrangement at least one yourself preferably has a detector field which extends essentially parallel to the laser beams and which is designed to use at least part of the detection spectrum which arises in the detection areas due to the excitation by the respective laser beam, via an assigned spectral filter, along a detection path which is assigned to the laser beam, preferably essentially parallel to the latter. and to detect the radiation guiding arrangement for emission radiation guided to the detector field, the spectral filter arrangement being matched to one another in terms of their spectral selectivity, the fluorescence markers in terms of their spectral absorption and emission selectivity, and the wavelengths of the laser beams such that at least four different fluorescence markers are independent using at least four laser beams resolved from each other after the respective gel electrophoresis path and the respective fluorescent marker are detectable.

Looking at the emission and absorption spectra of the fluorescent dyes usually used in gel electrophoresis, it appears at first glance to be completely impossible to detect four or more fluorescent markers after the respective fluorescent markers, i.e. to distinguish them from one another, for example with a spectral "residual crosstalk" less than 10%. One will easily find fluorescent marker combinations from two different fluorescent markers whose absorption spectra do not overlap and whose emission spectra do not overlap, so that a detection after the frequency marker is obviously possible. However, it was found that fluorescent markers with overlapping absorption spectra and fluorescence markers with overlapping emission spectra can also be detected in a fluorescence-marker-resolved manner. It was namely recognized that fluorescent markers with selectively excitable laser markers with overlapping emission spectra can be distinguished from one another on the basis of spatially and / or temporally separated laser beam excitation or / and on Basis of different wavelengths of the laser beams or / and on the basis of a phase-sensitive detection when excited with modulated laser radiation. It was also recognized that the laser radiation can be used to distinguish selectively or non-selectively excitable fluorescent markers with at least partially non-overlapping emission spectra on the basis of spectral / selective detection of the emission radiation and possibly on the basis of spatially separated laser beam excitation. It has been shown that, based on this teaching, a spectral "residual crosstalk" between the fluorescent dyes can be achieved in the detection less than 10%, namely even better than 0.1%.

The electrophoresis device according to the invention is preferably used for evaluating sequencing reactions on the basis of a sample-specific (clone-specific, in particular primer-specific) fluorescent dye marking. For this purpose, the four specific sequencing approaches belonging to a clone are separated and analyzed in four different gel electrophoresis lanes (gel tracks).

By means of the electrophoresis device according to the invention, however, it is also possible to evaluate sequencing reactions based on a base-specific fluorescent dye label, in which the four specific, differently labeled ones belonging to a clone

Sequencing approaches together in a gel electrophoresis lane

(Gel trace) can be separated and analyzed. In the case of mutually offset in the molecular propagation direction, different ones used for the base-specific labeling in relation to a clone

Laser beams assigned to dyes can then be used to compensate for the different separation distances for sequence determination, for example using software. The runtime can therefore be corrected in order to make a comparative one for the sequence determination

To enable analysis of the mobility of the sequencing fragments. In the

As a rule, it will suffice to correct the maturities after a linear one Approach (set of three in relation to the separation distances assuming constant fragment propagation speed along the gel electrophoresis lanes). Furthermore, the “mobility shifts” that inevitably occur can be compensated for as far as possible in a manner known per se.

The spectral filter and radiation guide arrangement assigned to a fluorescent dye can be interpreted in connection with the assigned detector field as a detection unit or detector unit assigned to this fluorescent dye, and if desired can actually be designed as a "unit that can be handled". It is preferred for the detection units that the intensity ratio of the light originating from the fluorescent dye belonging to the respective detection unit to the light originating from another fluorescent dye is 1: 0.2 or greater for a detection unit. The detection units for the fluorescence radiation to be detected preferably have an acceptance angle of approximately ± 20 ° in relation to an optical axis of the detection unit.

It is proposed that the wavelengths of the laser beams and / or spectral filters of the spectral filter arrangements be matched to at least four different fluorescence markers, the absorption maxima of which are at least 30 nm, preferably at least 50 nm, apart from each other under electrophoresis operating conditions or / and their Emission maxima are at least 30 nm apart, preferably at least 50 nm apart, under electrophoresis operating conditions.

It is extremely expedient if at least one spectral filter has a spectral characteristic in such a way that fluorescence radiation of the respectively assigned fluorescence marker, which arises due to the excitation of the respectively assigned laser beam, for a transmission Wavelength range around an emission maximum of the fluorescence spectrum of this fluorescence marker or for a transmission wavelength range adjacent to this fluorescence maximum is guided to the assigned detector field. In a further development it is proposed that at least one of the following conditions a) and b) is fulfilled with respect to at least one of the other fluorescent markers in order to enable the detection resolved after the fluorescent markers: a) the laser beam wavelength lies on a spectral side of an absorption spectrum of the other fluorescent markers beyond one from an absorption maximum to the laser beam wavelength down to at least one

Residual absorption amplitude, which corresponds to an at most low residual absorption, falling edge of the absorption spectrum, b) the transmission wavelength range lies on a spectral side of an emission spectrum of the other fluorescence marker beyond one from an emission maximum to the transmission wavelength range down to at least one residual emission amplitude, which is at most a low one Residual emission corresponds to falling edge of the emission spectrum.

The flank falling to the laser beam wavelength can be a flank falling to the longer wavelengths, and the flank falling to the transmission wavelength range can be a flank falling to shorter wavelengths.

Alternatively, it is possible for the edge falling to the laser beam wavelength to be an edge falling to shorter wavelengths and the edge falling to the pass wavelength range to be an edge falling to longer wavelengths.

It is generally proposed that the flank falls more steeply towards the laser beam wavelength or the transmission wavelength range than a flank of the spectrum lying on the other side of the maximum of the spectrum and falling with the distance from the maximum. At least one spectral filter and radiation guide arrangement can comprise a lens arrangement, preferably a gradient index lens field (possibly SELFOC lens field (SLA)), as the radiation guide arrangement. The gradient index field should be designed to map fluorescent light onto the assigned detector field along the relevant detection path alone or in conjunction with other optical components and can also be used as. "Linear gradient index field" are called. At least one spectral filter on the object side can be arranged on an object side of the radiation guide arrangement assigned to the gel electrophoresis tracks. Furthermore, at least one image-side spectral filter can also be arranged on the image side of the radiation guiding arrangement associated with the detector field. The radiation guide arrangement can also comprise further optical means, for example a diaphragm arrangement, in order to increase the optical or spectral selectivity.

At least one spectral filter is preferably designed as an interference filter or comprises an interference filter. For this purpose, it is specifically proposed that at least one spectral filter arrangement be designed as a combination of an absorption filter arrangement and an interference filter arrangement. For example, a spectral filter can include an absorption filter, possibly a colored glass filter, which serves as a carrier substrate for thin-film films of an associated interference filter.

The use of an interference filter may be surprising, since the spectral selectivity of such a filter depends on the angle of incidence of the emission radiation into the filter. Especially when using a radiation guide arrangement with a high numerical aperture, for example a corresponding gradient index lens field, in order to collect the fluorescence photons emitted by the fluorescence markers with high efficiency, the use of an interference filter appears in view of the desired spectral selectivity of the Filters extremely problematic. However, it was recognized that the interference filter, which defines a transmission range as a transmission wavelength range, which corresponds to a superimposition of transmission ranges of the interference filter for different light incidence angles in the interference filters, does not necessarily pass this transmission wavelength range alone, but with a corresponding arrangement in relation to the radiation guide arrangement defines the transmission wavelength range together with the radiation guide arrangement. A wavelength range can thus be defined as the transmission wavelength range, which includes all transmission ranges of the interference filter for all light incidence angles that occur according to an acceptance angle characteristic of the radiation guide arrangement. For example, a wavelength range can be defined as the transmission wavelength range, which comprises a transmission range of the interference filter for a light incidence angle of a light incidence angle of a = 0 ° and a transmission range of the interference filter for a light incidence angle corresponding to a maximum acceptance angle of the radiation guiding arrangement. If one takes into account such a, the acceptance angle I characteristic of the radiation guiding arrangement is taken into account by the transmission wavelength range in the design of the electrophoresis device, then there are many constructive possibilities in the design and specification of the spectral filter in relation to the Emission spectra of the

_ using fluorescent dyes, and spectral filter arrangements with comparatively high spectral selectivity can be created using interference filters in order to enable detection that resolves the fluorescent markers.

On the basis of the principles explained, the electrophoresis device according to the invention can be designed to detect at least seven markers from the following fluorescent markers: indodicarbocyanine CY 2, Alexa 488, fluorescein iso-thio-cyanate (FITC), 5-carboxy-rhodamine 6G, Alexa 532, tetramethyl-rhodamine-iso-thio-cyanate (TRITC), Indodicarbocyanin CY 3, Sulforhodamine 101 (TexasRed ™), Indodicarbocyanin CY 5, Indodicarbocyanin CY 5.5, IRD 700, Indodicarbocyanin CY 7, IRD 40 and IRD 800.

Specifically, at least the markers indodicarbocyanin CY 2, fluorescein isothio cyanate (FITC), 5-carboxy-rhodamine 6G, sulforhodamine 1 01 (TexasRed ™), indodicarbocyanine CY 5, indodicarbocyanine CY 5.5 and IRD 800 can be detected.

The electrophoresis device can be specially designed to detect at least three, preferably at least four, most preferably at least five, markers of fluorescent marker-dissolved and gel-electrophoresis-path-resolved simultaneously of the following fluorescent markers: indodicarbocyanine CY 2, Alexa 488, fluorescein iso -Thio- cγanate (FITC), 5-carboxy-rhodamine 6G, Alexa 532, tetramethyl-rhodamine-iso-thio-cyanate (TRITC), indodicarbocyanine CY 3, sulforhodamine 1 01 (TexasRed ™), indodicarbocyanine CY 5, indodicarbocyanine CY 5.5 , IRD 700, indodicarbocyanine CY 7, IRD 40 and IRD 800.

Specifically, it is suggested that at least the five markers indodicarbocyanine CY 2, 5-carboxy-rhodamine 6G, Sulforhodamine 101 (TexasRed ™), indodicarbocyanine CY 5.5 and IRD 800 or at least the four markers fluorescein-iso-thio-cyanate (FITC), sulforhodamine 101 (TexasRed ™), indodicarbocyanine CY 5.5 and IRD 800 or at least the four markers indodicarbocyanin CY 2, 5-carboxy-rhodamine 6G, indodicarbocyanin CY 5 and IRD 800 or at least the three markers fluorescein iso-thio-cyanate (FITC), Indodicarbocyanin CY 5 and IRD 800 can be detected at the same time as fluorescent marker-resolved and gel-electrophoretic-web-resolved.

When designing the spectral filter and when selecting the excitation wavelengths, there are basically many possibilities, which of depend on the available laser systems and the available spectral filters. Basic boundary conditions are only the selectivity of the excitation, the excitation efficiency achievable for a specific excitation wavelength, the selectivity of the detection and the detection efficiency achievable with a certain passband with respect to the spectral sensitivity of a detector used. In practice, however, other boundary conditions will also play an important role, namely the costs of the laser systems in terms of acquisition and maintenance and the costs for the acquisition of the spectral filters and detectors. In the following, some examples of laser systems and spectral filters that can be used are given in relation to various of the fluorescent markers mentioned, which are in no way restrictive, but are only intended to provide an indication. It is obvious, therefore, that in the case of lasers which can be tuned with regard to the wavelength, such as, for example, laser diodes, excitation wavelengths which deviate from the following information are also suitable within the excitation spectra which are generally quite wide. The excitation wavelengths mentioned therefore only stand for a possible wavelength range of, for example, approximately ± 10 to 20 nm on both spectral sides of the wavelength mentioned. The same applies to the pass bands of the spectral filter arrangements specified below. It is obvious that spectral filter arrangements with spectrally narrower passbands in the range of the passbands mentioned and / or radiation guiding arrangements with a smaller acceptance angle range can be used in any case if a lower detection efficiency (higher photon losses) is accepted. But one can also use spectral filter arrangements with larger pass bands or spectra I offset through I a ß ranges and / or radiation guiding arrangements with larger acceptance angle ranges depending on the emission spectra of the fluorescent markers, as long as the detection selectivity of the electrophoresis device as a whole remains preserved. Accordingly, the spectral limits of the passbands mentioned below only give an indication of the design of the spectral filter arrangements, and the passbands mentioned can be spectrally offset by, for example, approximately ± 10 to 20 nm or expanded to smaller or / and larger wavelengths. However, the data given below for possible excitation wavelengths, pass bands and maximum acceptance angles define preferred embodiments of an electrophoresis device according to the invention, with which excellent analysis results were achieved in electrophoresis operation.

For the detection of the fluorescence marker indodicarbocyanine CY 2, it is proposed to use a frequency-doubled (possibly diode-pumped) neodymium: YAG laser with an emission wavelength of approximately 473 nm. A spectral filter arrangement with a pass band of approximately 494 nm to approximately 424 nm can be assigned to this fluorescence marker. If different light angles of incidence a play a role in the spectral filter arrangement, then the spectral filter arrangement can have different light incidence angles a assigned transmission ranges from approximately 494 nm to approximately 524 nm for a = 0 ° and approximately 492 nm to approximately 514 nm for a maximum acceptance angle d have light incidence angles corresponding to the radiation guide arrangement (for example of approximately 20 °). The spectral filter arrangement may comprise an absorption short pass filter, an absorption guide pass filter and an interference band pass filter.

An argon ion laser with an emission wavelength of approximately 488 nm or a frequency-doubled (possibly diode-pumped) neodymium: YAG laser with an emission wavelength of approximately 473 nm can be used for the detection of the fluorescence marker fluorescein iso-thio-cyanate (FITC) become. A spectral filter arrangement with a pass range from approximately 505 nm to approximately 555 nm, possibly with, can be attached to this fluorescent marker Passage ranges of approximately 505 nm to approximately 555 nm for a = 0 ° and approximately 500 nm to approximately 555 nm for a light incidence angle corresponding to a maximum acceptance angle of the radiation guiding arrangement (for example of approximately 20 °) can be assigned to different light incidence angles a. The spectral filter arrangement can comprise an absorption short-pass filter, an absorption long-pass filter and an interference band-pass filter.

For the detection of the fluorescent marker 5-carboxy-rhodamine 6G, it is proposed to use a frequency-doubled (possibly diode-pumped) neodymium: YAG laser with an emission wavelength of approximately 532 nm. The spectral filter arrangement assigned to this fluorescence marker can have a pass band from approximately 555 nm to approximately 578 nm, possibly pass bands assigned to different light incidence angles a from approximately 555 nm to approximately 578 nm for a = 0 ° and approximately 535 nm to approximately Have 569 nm for a light incidence angle corresponding to a maximum acceptance angle of the radiation guide arrangement (for example of approximately 20 °). The spectral filter arrangement can comprise an absorption short-pass filter and an interference band-pass filter.

A helium-neon laser with an emission wavelength of approximately 594 nm can be used for the detection of the fluorescence marker Sulforhodamine 101 (TexasRed ™). A spectral filter arrangement with a transmission range of approximately 61 3 nm to approximately 651 nm, possibly with transmission ranges of approximately 61 3 nm to 651 nm for a = 0 ° and approximately 61 3 nm to approximately 636 nm for one assigned to different light incidence angles σ can be assigned to this fluorescence marker corresponding light incidence particles (for example of approximately 20 °) can be assigned to a maximum acceptance angle of the radiation guide arrangement. The spectral filter arrangement can comprise an absorption long-pass filter and an interference band-pass filter. A helium-neon laser with an emission wavelength of approximately 633 nm or a semiconductor laser with an emission wavelength of approximately 640 nm can be used for the detection of the fluorescence marker indodicarbocyanine CY 5. A spectral filter arrangement with a pass band of approximately 655 nm to approximately 690 nm, possibly with pass band ranges of approximately 655 nm to approximately 690 nm for a - 0 ° and approximately 650 nm to approximately 683 nm for one, assigned to different light incidence angles a can be attached to this fluorescence marker corresponding to a maximum acceptance angle of the radiation guide arrangement corresponding light incidence angle (for example of approximately 20 °). The spectral filter arrangement can comprise an absorption long-pass filter and an interference band-pass filter.

A semiconductor laser with an emission wavelength of approximately 660 nm or approximately 670 nm or approximately 675 nm can be used for the detection of the fluorescence marker indodicarbocyanine CY 5.5 or / and the fluorescence marker IRD 700. The spectral filter arrangement in question can have a transmission range from approximately 655 nm to approximately 690 nm, possibly transmission ranges assigned to different light incidence angles a from approximately 695 nm to approximately 745 nm for a = 0 ° and approximately 695 nm to approximately 730 nm for a maximum acceptance angle have the light incidence angle corresponding to the radiation guide arrangement (for example of approximately 20 ° C.). The spectral filter arrangement can comprise an absorption long-pass filter and an interference band-pass filter.

A semiconductor laser with an emission wavelength of approximately 775 nm or of approximately 780 nm can be used for the detection of the fluorescence marker IRD 800. A spectral filter arrangement with a transmission range of approximately 795 nm to approximately 790 nm, possibly with transmission ranges of approximately 795 nm to approximately 835 nm for a = 0 ° and approximately 790 nm to approximately 830 nm for one assigned to different light incidence angles, can be assigned to this fluorescence marker a maximum Acceptance angles of the radiation guiding arrangement can be assigned corresponding light incidence angles (for example of approximately 20 °). The spectral filter arrangement can comprise an absorption long-pass filter and an interference band-pass filter.

In the electrophoresis device according to the invention, measures are preferably taken to reduce the resolution of the detection in relation to the gel electrophoresis path or / and in relation to the fluorescence markers and / or interference radiation which may impair the signal-to-noise ratio, possibly fluorescent light. Background radiation and / or scattered laser radiation. For example, at least one of the laser beams can be linearly polarized with a polarization vector, which is oriented according to an input-side light guiding direction of the spectral filter and radiation guiding arrangement, possibly at least approximately parallel to this. This minimizes the scattered light emitted in the direction of the detectors, since with the elastic (Rayleigh scattering the scattered light is preferably emitted into a plane perpendicular to the polarization vector of the incident light. Such a direction of polarization can be achieved by arranging the laser appropriately or / and by Use of appropriate optical components, for example polarizers or / and Λ / 2 plates, can be achieved.

With regard to the gel electrophoresis lanes, it is preferred that at least 20, preferably at least 40 and particularly preferably at least 80 (for example 1 20 or even 1 92 or 384 lanes) can be evaluated in parallel. The gel electrophoresis sheets can run in a preferably plate-shaped gel matrix which is arranged between glass plates made of floated borosilicate glass. Because of its high planarity, so-called floated glass avoids multiple reflections of the laser beams running between the glass plates, so that an increase in the scattered light and fluorescent light background due to multiple penetration of the laser radiation into the glass plates is avoided. Borosilicate glass is available in qualities with low intrinsic fluorescence, such as floated borosilicate glass, for example under the trade name Borofloat from Schott in Jena (Germany).

For the coupling of the laser beams into the gel matrix, it is particularly expedient to use at least one (preferably a common) coupling plate which is essentially parallel to the direction of laser beam propagation, the polished entry and / or exit surfaces for the respective laser beam and a refractive index adapted to the gel matrix having.

In order to avoid premature degradation of the fluorescent dyes and thus possibly reduced signal levels, the spatially offset laser beams having different wavelengths can be offset from one another in such a way that the laser radiation wavelength decreases in the molecular propagation direction from laser beam to laser beam. Accordingly, the fluorescent markers first pass through the laser beams with a longer wavelength before they reach the laser beams with a shorter wavelength. A degradation of fluorescent dyes to be excited by laser radiation with a longer wavelength by laser radiation with a shorter wavelength before the detection of the fluorescent marker in question is accordingly avoided.

The electrophoresis device described can be used for the analysis of nucleic acids, for example in such a way that products of nucleic acid sequencing reactions labeled with at least four different fluorescent markers are separated together in one lane according to their size and analyzed independently of one another. The separation can be carried out by electrophoresis in a denaturing gel. A parallel separation is preferably carried out in several lanes, each with at least four differently labeled products contain. In this way, using conventional gels, the evaluation of z. B. 1,000-1,200 bases per sequencing reaction, and a simultaneous separation of four sequencing reactions in one lane, using a plate gel with 1 92 tracks, more than 100,000 bases, eg. B. 1 55,000 bases can be determined on a single gel. In the case of gels with a larger number of tracks, as mentioned above, more bases can be determined accordingly. For example, based on doubling the track density (384 tracks on a single gel), more than 200,000 bases, e.g. B. 300,000 to 384,000 bases, can be determined on a single gel.

The gel electrophoresis device used for the analysis and used as the sequencing device is preferably largely automated (preferably especially with regard to the sequence determination) and preferably has a separate excitation laser and a separate detection unit for each fluorescent dye. To increase the (spectral) selectivity, other optical means, for example diaphragms, are used if desired in addition to the spectral filters mentioned.

The fluorescent markers can be selected, for example, such that the absorption maxima are at least 30 nm apart, preferably at least 50 nm apart, under the measurement conditions. Furthermore, the fluorescent markers can be selected such that the emission maxima are at least 30 nm apart, preferably at least 50 nm apart, under the measurement conditions.

The above-mentioned fluorescent dyes can be used as fluorescent dyes, for example the dye combination: fluorescein, TexasRed ™, CY 5.5 and IRD 800, or for example the dye combination: 5-carboxy-rhodamine 6G, TexasRed ™, CY 5.5, IRD 800 and possibly CY 2 , or for example the dye combination: 5- Carboxy-Rhodamine 6G, TexasRed ™, CY 5.5, CY 7 and possibly CY 2 or / and IRD 800.

The sequencing products separated together in one lane can originate from a sequencing reaction carried out in a single vessel. At least four differently labeled primers can be used for the sequencing reactions. The sequencing reaction can be carried out as a quadruplex reaction, to the tandem duplex reaction, as a sequence gap filling reaction or as a competitive tandem duplex reaction.

With regard to such a use of the electrophoresis device according to the invention, the invention further relates to a reagent kit for sequencing nucleic acids which contains at least four different fluorescent dye labels, each label being provided for determining a different sequence. The kit can contain at least four different primers, each with a different label.

If one wants to make optimal use of the length of the available gel electrophoresis tracks to maximize the separation distance in view of a high resolution and long reading lengths and to query fluorescence markers by several spatially offset laser beams, then it is desirable to determine the distance between the laser beams to be kept relatively small in the direction of molecular propagation, so that separation distances of the same order of magnitude are available for the fluorescent markers assigned to the laser beams. For this purpose, it is proposed according to a second aspect of the invention that the spectral filter and radiation guiding arrangements redirect the emission radiation emanating from the excited flow mark to reception surfaces of the detector field assigned to the respective laser beam, which at least in relation to the direction of molecular propagation are arranged approximately orthogonally. This enables a compact design of the detection unit in the direction of molecular propagation, so that accordingly the laser beams also cut the gel electrophoresis tracks at a comparatively small distance from one another.

This inventive concept is also independent of the maximization of the separation distance in the case. the simultaneous separation of a plurality of fluorescent dyes of interest, so that, according to the second aspect, there is generally provided an electrophoresis device for analyzing fluorescence marker-labeled molecules, in particular biological molecules, which comprises: a plurality of substantially parallel gels running parallel to one another and in the length of a molecular propagation direction Electrophoresis webs, an energy source for applying an electrical potential to an initial region and an end region of the gel electrophoresis webs, a laser arrangement for excitation of the fluorescence markers associated with the molecules, the laser arrangement in electrophoresis mode generating laser radiation which is essentially transverse to at least one The laser beam extending in the direction of molecular propagation cuts at least several of the gel electrophoresis tracks in order to detect the fluorescence markers of the Mo present there in a detection area of the respective gel electrophoresis track excite a detection arrangement for detecting emission radiation emanating from the excited fluorescent markers, wherein the detection arrangement has at least one detector field, which extends preferably essentially parallel to the at least one laser beam, and which is designed for this purpose, along a path assigned to the laser beam, preferably essentially to the latter parallel detection path to detect at least a part of the emission radiation which arises in the detection areas due to the laser beam excitation and is guided to the detector field via an assigned spectral filter and radiation guide arrangement, the spectral filter and Radiation guidance arrangement deflects the emission radiation emanating from the excited fluorescence marker onto receiving surfaces or receiving surface sections of the detector field, which are arranged at least approximately orthogonally to the direction of molecular propagation.

It is particularly expedient to arrange the (respective) detector field on a carrier board which is arranged at least approximately orthogonally to the direction of molecular propagation. As already indicated, a plurality of detector fields, preferably of different wavelengths, assigned to a plurality of laser beams in succession in the molecular propagation direction can be provided, each with receiving surfaces or receiving surface sections arranged at least approximately orthogonally orthogonally to the molecular propagation direction, which are preferably each arranged on a separate carrier board. The spacing of the carrier board is primarily determined by the thickness of any components arranged on the carrier board, possibly the detector fields, or a dimension of the radiation guide arrangement, but not by the size of the individual detector fields, so that the carrier boards can be arranged at a short distance from one another.

The spectral filter and radiation guide arrangement can comprise at least one deflection prism arrangement which is assigned to a respective detector field and extends essentially parallel to this detector field, optionally a deflection prism which is continuous (in the longitudinal direction). The deflection prism arrangement can be arranged on an output side of a lens arrangement, possibly a gradient index lens field, of the spectral filter and radiation guide arrangement.

To adapt an optical path on the image side between a / the lens arrangement, possibly a / the gradient index lens field, the spectral filter and radiation guide arrangement on the one hand and the On the other hand, if the receiving surfaces are at an image-side focal length of the lens arrangement, the deflection prism arrangement can be formed from a highly refractive optical material, possibly flint glass SF4.

The deflection prism arrangement is preferably part of an integral optical module which comprises a / the lens arrangement and at least one spectral filter. For example, the deflection prism arrangement, the lens arrangement designed as a gradient index lens field, the at least one spectral filter and, if desired, a carrier rail for producing the integral optical module can be glued together. At least one spectral filter is stuck on the optical index side of the gradient index field (possibly between the gradient index lens field and the deflecting prism arrangement) on the optical index side of the gradient index field (or between the gradient index lens field and the deflection prism arrangement).

At least one spectral filter of the optical module can be designed as an interference filter or can comprise an interference filter. At least one spectral filter is preferably designed as a combination of an absorption filter and an interference filter. For this purpose, it is proposed that at least one spectral filter of the optical module comprises an absorption filter, possibly a colored glass filter, which serves as a carrier substrate for thin-film films of an associated interference filter.

To adjust an optical path in a module carrier, the optical module can be arranged so as to be displaceable in a direction substantially orthogonal to the laser beam sections intersecting the gel electrophoresis tracks and to the direction of molecular propagation.

A preferred embodiment is characterized in that a plurality of detector fields, a plurality of each one of the

Optical modules assigned to detector fields as well as detection electronics assigned to the detector fields in a detector module with a common housing are integrated, the detector module being manageable as a unit and preferably by shifting in a laser beam section intersecting the gel electrophoresis paths and essentially orthogonal to the direction of molecular propagation, a common adjustment of the object-side optical path lengths assigned to all optical modules of the detector module is possible ,

With regard to the basic structure of an electrophoresis device, for example the electrophoresis devices according to the first or second aspect of the invention, there are basically many possibilities. With regard to a compact and simple structure of the electrophoresis device and / or with regard to a high directional stability of the laser beams (in this respect, for example, high demands have to be made on DNA sequencing), it is proposed according to a third aspect that the laser arrangement has at least one Has a laser with an optical laser resonator, which is arranged at least approximately parallel to the detector fields in relation to a resonator axis which characterizes the beam path of the laser beam emerging from the resonator, the laser beam emerging from the laser resonator being deflected by means of a beam guiding arrangement, so that a beam which runs transversely to the molecular propagation direction Laser beam section cuts the gel electrophoresis sheets. Alternatively or additionally, it is proposed that the laser arrangement have at least two lasers with a respective laser resonator, which are each arranged at an angle to the detector fields, preferably at least approximately orthogonally to the detector fields, with respect to a resonator axis that characterizes the beam path of the laser beam emerging from the respective resonator are arranged, the laser beam emerging from the respective laser resonator being deflected by means of a beam guidance arrangement, so that a laser beam section running transversely to the direction of molecular propagation intersects the gel electrophoresis tracks and the laser resonators are covered on the resonator axes, at least approximately in accordance with the offset of the gel-electrophoresis paths, the laser beam sections intersecting each other in the molecular propagation direction are offset from one another and have different lateral spacing from the gel electrophoresis tracks.

Since the specified structure of the electrophoresis device can not only be advantageous for the electrophoresis device according to the first and / or according to the second aspect, the invention according to the third aspect generally provides an electrophoresis device for analyzing fluorescence marker-labeled molecules, in particular biological molecules, comprising: a plurality of gel electrophoresis sheets substantially parallel to one another and along a molecular propagation direction, an energy source for applying an electrical potential to a start area and an end area of the gel electrophoresis sheets, a laser arrangement for excitation a plurality of different fluorescent markers assigned to the molecules, the laser arrangement in electrophoresis mode generating laser radiation which is essentially transverse to the direction of molecular propagation and essentially parallel to one another and extends in the direction of molecular propagation Laser beams of different wavelengths offset against one another intersect at least several of the gel electrophoresis tracks, in order to excite fluorescence markers of the molecules present in a detection area of the respective gel electrophoresis track, a detection arrangement for gel-electrophoresis-track-resolved and fluorescence-marker-resolved detection emission radiation emitted by the excited fluorescent markers, the detection arrangement having a plurality of detector fields which are offset from one another in the direction of molecular propagation and preferably extend essentially parallel to the laser beams, each of which is associated with a specific one of the laser beams and is designed for this purpose, along one associated with the respective laser beam , preferably too This essentially parallel detection path detects at least a part of the emission radiation which is generated in the detection areas due to the excitation by the respective laser beam and is spatially resolved via a respectively assigned spectral filter and radiation guide arrangement to the detector field, the laser arrangement at least one Has a laser with an optical laser resonator, which is arranged at least approximately parallel to the detector fields with respect to a resonator axis that characterizes the beam path of the laser beam emerging from the resonator, the laser beam means that originate from the laser being of its beam guide arrangement is deflected so that a laser beam section running transversely to the molecular propagation direction intersects the gel electrophoresis tracks.

A plurality of lasers with at least approximately parallel resonators with respect to a respective resonator axis to the detector fields can be provided, the laser resonators with respect to the resonator axes being at least approximately offset from one another in the direction of molecular propagation in accordance with the offset of the laser beam sections cutting the gel electrophoresis tracks.

Furthermore, according to the third aspect, the invention provides an electrophoresis device for analyzing fluorescence-labeled molecules, in particular biological molecules, which comprises: a plurality of gel electrophoresis devices which run essentially parallel to one another and along a direction of molecular propagation. Webs, an energy source for applying an electrical potential to an initial region and an end region of the gel electrophoresis webs, a laser arrangement for exciting a plurality of different fluorescent markers assigned to the molecules, the laser arrangement in the electrophoresis mode generating laser radiation which is considered to be essentially transverse to the direction of molecular propagation and essentially parallel to each other Laser beams of different wavelengths running and mutually offset in the direction of molecular propagation intersect at least several of the gel electrophoresis tracks, in order to excite fluorescence markers associated with the molecules present in a detection area of the respective gel electrophoresis track, a detection arrangement for the gel electrophoresis track-resolved and fluorescence markers Detected emission radiation emitted by the excited fluorescent markers, the detection arrangement having a plurality of detector fields which are offset from one another in the direction of molecular propagation and preferably extend essentially parallel to the laser beams, each of which is assigned to a specific one of the laser beams and is designed for this purpose, along one of the respective ones Laser beam associated, preferably to this substantially parallel detection path at least a part of the detection areas due to the excitation to detect locally generated emission radiation generated by the respective laser beam via a respectively assigned spectral filter and radiation guide arrangement to the detector field, the laser arrangement having at least two lasers with a respective laser resonator, each of which relates to the beam path from the respective one The resonator axis characterizing the resonator emerging laser beam at an angle. are arranged to the detector fields, preferably are arranged at least approximately orthogonally to the detector fields, the laser beam emerging from the respective laser resonator being deflected by means of a beam guidance arrangement, so that a laser beam section running transversely to the direction of molecular propagation intersects the gel electrophoresis paths and the laser resonators are related on the resonator axes are at least approximately offset according to the offset of the laser beam sections intersecting the gel electrophoresis tracks and have different lateral spacing from the gel electrophoresis tracks. For this purpose, it is further proposed that the resonator axes lie at least approximately in a respective plane orthogonal to the direction of molecular propagation.

A particularly preferred embodiment is characterized by a stepped optical bench arranged to the side of the gel electrophoresis tracks with steps arranged at an angle / to the direction of molecular propagation, preferably at least approximate or orthogonal to the direction of molecular propagation, with increasing lateral distance from the gel electrophoresis paths opposite to the direction of molecular propagation, at least approximately in accordance with the offset of the gel electrophoresis pathways, increasing and assigned to a respective one of the lasers for holding a respective one assigned to the laser Beam guiding arrangement and / or the laser resonator with its resonator axis extending at least approximately along the stage.

With regard to the configuration of the beam guidance arrangement, various variants are conceivable. It is proposed, for example, that the respective beam guidance arrangement be an optical fiber, preferably a single-mode fiber, and / or at least one deflecting mirror or / and at least one optical element for focusing the laser beam and / or adjusting a laser beam waist with respect to the gel electrophoresis paths, if necessary has an optical lens, an optical lens or an optical telescope, and / or an optical element for conditioning, possibly spatial filtering, the laser beam. In this context, it is preferred that at least one deflecting mirror and / or the optical element for adjusting the beam path of the respective laser beam is adjustable, preferably adjustable by motor.

For this purpose, it is further developed that a deflecting mirror, from which the laser beam section cutting the gel electrophoresis tracks goes out, at least approximately in a direction orthogonal to the molecular propagation direction and the laser beam section, preferably at least approximately along the respective stage of the optical bench, for adjustment of the laser beam section cutting the gel electrophoresis tracks or / and that at least one optical element at least approximately in the direction of molecular propagation is displaceable for the adjustment of the laser beam section intersecting the gel electrophoresis tracks.

The use of an optical fiber, preferably a single-mode fiber, in a beam guiding arrangement is also important independently of the rest of the design of an electrophoresis device, since the optical radiation can easily condition and / or guide the laser radiation to the gel electrophoresis tracks can be achieved. Regardless of the optical arrangement used for this purpose, the conditioning of the laser radiation is also of interest with regard to a high triggering capacity and a minimization of the signal-to-noise ratio of the / the electrophoresis device. According to a fourth aspect, the invention therefore provides an electrophoresis device for analyzing fluorescence-labeled molecules, in particular biological molecules, which comprises: a plurality of gel electrophoresis tracks which run essentially parallel to one another and along a direction of molecular propagation, an energy source for applying an electrical potential to an initial region and an end region of the gel electrophoresis tracks, a laser arrangement for exciting a plurality of fluorescence markers assigned to the molecules, the laser arrangement in the electrophoresis mode generating laser radiation which acts as at least one laser beam which runs essentially transversely to the molecule propagation direction cuts at least several of the gel electrophoresis tracks in order to excite fluorescence markers of the molecules present in a detection area of the respective gel electrophoresis track, a detection arrangement for gel electrophoresis path-resolved and fluorescence marker-resolved detection of emission radiation emanating from the excited fluorescence markers, wherein the detection arrangement has at least one detector field which extends essentially parallel to the laser beams and is designed for this purpose along a path assigned to the laser beam , preferably to detect at least a portion of the emission radiation generated in the detection areas due to the excitation by the associated laser beam and guided to the detector field via an assigned spectral filter and radiation guide arrangement, with the laser arrangement having at least one laser whose laser beam is in an optical fiber, preferably single-mode fiber, is coupled in to guide the laser beam to the gel electrophoresis tracks or / and to condition the laser beam, or / u nd wherein the laser arrangement has at least one laser, the laser beam of which is guided through at least one optical element for conditioning, possibly spatial filtering, the laser beam.

For this purpose, it is further developed that the laser beam, which may have a non-Gaussian profile beforehand, is conditioned in such a way that it is at least approximately a Gaussian profile and / or a reduced divergence or / and a reduced beam waist diameter or / and a reduced one , with the product of a divergence angle describing the divergence and the beam waist diameter increasing collimation factor, possibly so-called M ² factor.

By using laser beams conditioned in this way or having such a quality from the outset, the selectivity of the electrophoresis device is maximized, since this is essentially determined by the width of the exciting laser beam in the direction of molecular propagation. The area "illuminated" in the gel is a kind of target line for the migrating molecules, possibly DNA fragments. For a good one Resolution, possibly temporal or vertical resolution, the illuminated area must be as narrow as possible. Furthermore, the areas illuminated in the individual cut gel electrophoresis lanes should differ as little as possible in their width in the direction of molecular propagation in order to achieve comparable results for all lanes. For this purpose, it is also specifically proposed that at least one of the laser beams is guided and, if necessary, focused in such a way that a laser beam region of minimal laser beam diameter, possibly one / the beam waist, along the assigned detector field, for example in the region of a central one of the gel electrophoresis cut by the laser beam. Tracks is arranged.

The resolution achieved with an electrophoresis device, possibly the electrophoresis device according to one of the mentioned aspects of the invention, and generally the quality of the measurement results depend to a large extent on an adjustment of the various components and in particular the course of the laser beams (generally: at least one Laser beam) in relation to assigned gel electrophoresis tracks. For this purpose, according to a fifth aspect of the invention, an adjustment method for adjusting the course of at least one laser beam with respect to associated gel electrophoresis paths of an electrophoresis device which has at least one excitation laser for generating the laser beam and at least one detector field for recording emission radiation emanating from excited fluorescence markers , provided, in which, according to the invention, the adjustment is carried out on the basis of detection signals recorded by means of a detector field assigned to the (respective) laser beam, which represent a background radiation induced by the laser beam, possibly background fluorescence and / or scattered light.

For this purpose, it is further developed that in the course of the adjustment at least one optical element associated with the laser beam, possibly a lens, an objective, a telescope or a deflecting mirror, is used for the recording a background radiation profile is adjusted, and the optical element is then set such that a background radiation is subsequently detected at least approximately according to a maximum and / or a center of the profile.

It is particularly preferred that first a laser beam path intersecting the gel electrophoresis paths and then a laser beam path at least approximately orthogonal to the gel electrophoresis paths or / and a laser beam path at least approximately parallel to the assigned detector field is set.

A laser beam path intersecting the gel electrophoresis tracks can expediently be set particularly advantageously on the basis of detection signals picked up by at least one detector field section at one end, preferably at the laser beam coupling point into a end of the detector field having the gel electrophoresis tracks and a laser beam path that is at least approximately orthogonal to the gel electrophoresis paths or / and a laser beam path that is at least approximately parallel to the detector field can be set particularly expediently on the basis of detection signals recorded by at least one detector field section at the other end of the detector field. This applies on the condition that there is no or only a slight degree of freedom with regard to the parallelism of the laser beam to a plane defined by the gel electrophoresis paths, possibly the gel matrix. If there is a degree of freedom that requires adjustment in this regard, this parallelism should first be set, for example by taking appropriate structural precautions of the gel electrophoresis device.

The adjustment can be carried out by a motor and preferably automatically or automatically if the gel electrophoresis device is equipped accordingly. In gel electrophoresis, so-called thermostatting plates are often used, which serve to keep the gel temperature as constant as possible in the direction perpendicular to the wall direction (direction of molecular propagation) of the molecules, i.e. to ensure a uniform temperature distribution across the direction of molecular propagation. At least one such thermostat plate, as described for example in DE 30 46 729 C2 or in the article "Improvements of DNA Sequencing Gels" by H. Garoff and W. Ansorge, Analytical Biochemistry 1 15, 450-457 (1 991) , can also be used advantageously in the electrophoresis devices according to the aspects of the invention mentioned.

It has now been found that it can be advantageous to use the thermostat plate in the gel (in the gel matrix) to set a temperature gradient in the direction of molecular propagation and / or in a direction associated with a thickness of the gel matrix and essentially orthogonal to the thermostat plate, in the case of a thermostat plate which for the thermostatting of a heat exchanger medium (in general water) is flowed through (approximately like the thermostatic plate known from DE 30 46 729 C2 or the article mentioned), preferably in the state of continuous flow through the thermostatic plate with the heat exchanger medium.

In general, according to a sixth aspect, the invention provides an electrophoresis device for analyzing labeled molecules, in particular biological molecules, which comprises: a plurality of gel electrophoresis tracks which run essentially parallel to one another and along a direction of molecular propagation, which run in a gel matrix, possibly a plate gel, at least one thermostatic plate which is in heat exchange connection with a gel matrix surface, an energy source for applying an electrical potential to a start region and an end region of the gel electrophoresis sheets, the thermostatic plate being suitable for this purpose . Establish and / or maintain a uniform temperature distribution in the gel matrix in the gel electrophoresis - path-cutting direction to the direction of molecular propagation, wherein the at least one thermostatic plate is further suitable for a temperature gradient in the direction of molecular propagation in the gel matrix and / or in a thickness associated with the gel matrix, which is essentially orthogonal to the thermostatic plate, wherein in the case of a thermostatic plate through which a heat exchanger means flows, this is preferably suitable for setting the temperature gradient in question in the state of continuous flow through the thermostatic plate with the heat exchanger means. Alternatively or additionally, the at least one thermostatic plate can be suitable for setting a temperature profile with a local temperature increase or a local temperature decrease in at least one transverse region of the gel matrix, in the case of the thermostatic plate through which a heat exchanger medium flows, preferably in the state of continuous flow.

There are no restrictions with regard to the operating principle of the thermostat plate; this is in particular not limited to the use of heat exchanger means flowing through the plate. For example, direct thermostatting of the plate by means of electrical means (e.g. heating elements, Peltier elements and the like) can be considered.

It is proposed that two thermostatic plates are provided, between which the gel matrix is arranged. A temperature profile can preferably be set with a temperature that decreases in the direction of molecular propagation, preferably in such a way that the temperature along the gel electrophoresis tracks decreases by several degrees (for example 2-10 °). Since the viscosity of the gel matrix decreases by approximately 2.4% per degree and a higher viscosity the mobility of the molecular bands in the gel Reduced accordingly, the decrease in temperature in the direction of molecular propagation causes the leading edge of each band to move from an area of higher mobility to an area of lower mobility. The result of this is that the bands moving under the influence of the electric field through the gel become sharper towards the detection region or at least diverge less, ie the band diffusion normally occurring during electrophoresis is counteracted at least to a certain extent. This "focusing effect" leads to a higher resolution and, in the case of the analysis of products of sequencing reactions, to longer sequence reading lengths.

Under certain circumstances, however, it can also be expedient to set a temperature profile with a temperature increasing in the direction of molecular propagation, for which the thermostatic plate is preferably suitable. There is a particularly high degree of flexibility when the plate allows an optional setting of different temperature profiles, for example one with increasing temperature in the direction of molecular propagation and one with decreasing temperature in the direction of molecular propagation.

Alternatively or additionally, based on the direction assigned to the thickness of the gel matrix, a temperature profile with a temperature maximum lying in a central region of the gel matrix can be adjustable. Due to the increasing mobility towards the central area, this leads to a concentration of the molecules in the central area of the gel matrix, which also counteracts a divergence of the band and results in a kind of focusing with higher resolution and longer sequence reading lengths. One explanation for this could be that there are more defined migration conditions for the molecules in the middle area of the gel opposite edge areas of the gel adjacent to the surfaces delimiting the gel. This concentration in the middle range is also with regard to a high excitation and Detection yield by a laser beam having, for example, a Gaussian beam profile is advantageous. In principle, however, the inventive proposal relating to the thermostat plate is independent of the type of marking of the molecules and of the type of detection of the marked molecules. For adjusting the temperature profile with a lying in a central region of the gel matrix maximum temperature it can _'be particularly expedient to arrange the gel matrix between two Thermostatisierplatten.

The thermostatic plate can be suitable for setting a local temperature increase in a transverse area of the gel matrix corresponding to an initial area. If a laser arrangement is provided for exciting a plurality of fluorescence markers assigned to the molecules, which generates laser radiation in electrophoresis operation and which cuts at least one of the gel electrophoresis tracks as at least one laser beam which runs essentially transversely to the direction of molecular propagation in order to detect the respective gel areas in a detection area. To excite fluorescence markers associated with the electrophoresis pathway of the molecules present there, the detectability of the molecules marked with fluorescence markers can be improved by setting a local temperature increase in a transverse region including the respective detection region. The thermostatic plate is preferably suitable for this. The temperature increase in the transverse region mentioned leads to a broadening of the fluorescence marker bands and to an increase in the band spacing, as a result of which the bands can be “scanned” better by the regularly spatially narrowly delimited laser beam. The achievable sequence reading length is not significantly deteriorated if, at least over a larger part of the gel electrophoresis paths, the temperature down to the respective detection area is lower than in the said transverse area. When operating an electrophoresis device with a thermostating plate through which a heat exchanger means can flow, it was surprisingly found that a significant improvement in the measurement results with regard to reading length and reliability could be achieved in that the flow of the heat exchanger means (heat exchange medium, possibly water) through the preheated by the heat exchanger means Thermostat plate was interrupted after reaching a predetermined gel temperature and / or a predetermined gel temperature distribution. Accordingly, in a seventh aspect, the invention provides a method of operating an electrophoresis device for analyzing labeled molecules, particularly biological molecules, comprising: a plurality of substantially parallel gel electrophoresis tracks in a gel matrix that are parallel to one another in the direction of molecular propagation , possibly a plate gel, run at least one thermostating plate which is in heat exchange connection with a planar gel matrix surface and through which a heat exchange medium can flow, and which is suitable for producing and / or maintaining a uniform temperature distribution in the gel matrix in the crosswise direction of the molecular propagation path that intersects the gel electrophoresis paths , an energy source for applying an electrical potential to a start region and an end region of the gel electrophoresis sheets. According to the invention, the method has the step of interrupting the flow of the heat exchanger means through the thermostat plate preheated by the heat exchanger means after a predetermined / predeterminable gel temperature or / and a predetermined / predeterminable gel temperature distribution has been reached.

According to the invention, the thermostatic plate can be used in a known manner to ensure an even temperature distribution in

Set and maintain the transverse direction to the molecular propagation direction. After preheating the thermostat plate (also under known as thermostatic plate or thermoplate), however, the heat exchanger medium supply is interrupted, for example a water circuit is interrupted by a thermostated water bath through the plate. This results in positive effects on the electrophoresis analysis, on the one hand, by preventing vibrations that are induced by the circulating heat exchanger means and, above all, affect the glass surfaces of the thermostatic plates. Avoiding these small vibrations is particularly important insofar as, in the case of laser-assisted fluorescence marker detection, these vibrations are transmitted to the laser beam running through the gel layer, grazing the surface of the thermostat plate or even penetrating into the adjacent glass plate of the thermostat plate. For example, since longer DNA fragments in the gel only provide very narrow bands in the direction of molecular propagation (approximately 0.1 mm and less), such vibrations of the laser beam around the bands can reduce the resolution for longer DNA fragments and thus limit the sequence reading length.

On the other hand, a positive effect results from the fact that after the interruption of the heat exchanger medium flow, the uniform

Temperature first to some extent by those developing in the adjacent gel layer during electrophoresis

Joule heat is maintained. Because of a

Heat exchange, in particular by convection (convection of the heat exchange medium in the thermostatting plate and / or the surrounding air in a housing of the electrophoresis device that accommodates the thermostating plate with the gel), will after a time result in a temperature profile in the gel that has the positive effects mentioned, such as focusing of molecular bands. So with a vertical arrangement of the gel matrix and the

Thermostat plate with vertical direction of molecular spread from top to bottom a higher temperature in an upper area of the gel matrix and set a lower temperature in a lower region of the gel matrix, the thermostat plate still ensuring that uniform temperature conditions are maintained in the direction transverse to the direction of molecular propagation (and to the electrophoresis field). Due to the temperature distribution in the vertical direction which occurs as a result of the interruption of the heat exchanger medium circuit, the described focusing effect on the molecular bands is achieved by influencing the viscosity of the water buffer in the gel and thus the mobility of the bands in the gel.

In the above-mentioned method for operating an electrophoresis device, it may also be expedient that two thermostatting plates are provided, between which the gel matrix is arranged. Depending on the design and structure of the electrophoresis device, after interrupting the flow in the gel matrix there will be a temperature gradient in the direction of molecular propagation (preferably according to a temperature profile with a decreasing temperature in the direction of molecular propagation) or / and in a direction associated with a thickness of the gel matrix and essentially orthogonal to the thermostatic plate (preferably according to a temperature profile assigned to a direction assigned to the thickness of the gel matrix with a temperature maximum lying in a central region of the gel matrix). It is also possible that, with an appropriate arrangement of the thermostat plate, a temperature profile is set with increasing temperature in the direction of molecular propagation, which can also be expedient. Furthermore, a temperature profile with a local temperature increase or a local temperature decrease can be established in at least one transverse region of the gel matrix. In order to achieve such a temperature profile, the heat radiation from the gel matrix can be specifically influenced by shielding means, for example at least one shielding plate, for example by a temperature increase in a transverse area of the gel matrix corresponding to an initial area or / and in the case of laser-assisted fluorescence marker detection with regard to the detectability - to be obtained in a transverse area including the detection areas of the gel electrophoresis tracks. In the latter case, it is advisable to design and arrange a detector unit that absorbs the fluorescent radiation in such a way that it provides for the appropriate shielding of the gel matrix and thus - as a result of less heat radiation from the gel matrix in the transverse region mentioned - for setting the temperature increase in the transverse region ,

The operating method according to the invention can advantageously be used in the electrophoresis devices or methods according to the first to fifth aspects of the invention.

The invention and its various aspects are explained in more detail below with the aid of several exemplary embodiments with reference to the attached figures and in the text or tables included in the figures.

Fig. 1 shows schematically the structure of an embodiment of an electrophoresis device according to the invention.

FIG. 2 shows a detector module of the device of FIG. 1 in one

View corresponding to a top view of input sides of five optics and detector field modules of the detector module arranged one above the other.

FIG. 3 shows an optics and detector field module according to FIG. 2 in one

Exploded view with a linear detector field and a spectral filter and radiation guide arrangement formed by a gradient index lens field and a spectral filter, which can be designed as an integral optical module. Fig. 4 shows a horizontal section through an electrophoresis

1 according to an embodiment variant in the vertical region of a spectral filter and radiation guiding arrangement designed as an integral optical module with a schematic indication of a linear detector field formed by a series of photodiodes with assigned control and evaluation electronics.

Fig. 5 shows schematically the beam path of the optical module of the

4 by a so-called notched plate, a first optical spectral filter, the gradient index lens field, a second optical spectral filter, a deflection prism on the photodiode field.

FIG. 6 shows a preferred embodiment of the thermal plate (thermostatic plate) shown schematically in FIG. 4.

FIG. 7 shows a diagram which shows the spectral sensitivity of the linear detector field formed from silicon photodiodes

Function of the wavelength indicates.

Fig. 8 shows nominated absorption and emission spectra of the

Electrophoresis using an electrophoresis device according to the invention in the case of a simultaneous

Analysis of molecules labeled with four different fluorescent dyes, for example products of nucleic acid sequencing reactions, preferably using four fluorescent dyes (fluorescent markers, fluorochromes), FITC, TexasRed ™, CY 5.5 and _. IRD 800 with

Specification of the preferred to stimulate the particular Fluorescent dye used excitation wavelength and the preferred type of laser used.

9 shows the chemical structure of those preferably used with an electrophoresis device according to the invention

Fluorescent dyes.

10 specifies one preferred for the detection of the

Fluorescent dye CY 2 used spectral filter and radiation guide arrangement with regard to a preferred spectral passband and a preferred structure of the spectral filter arrangement, wherein in a table (Fig.10a) a transmission of 50% corresponding edge wavelengths of the spectral filter arrangement and data for the transmission of the spectral filter arrangement are given and in the diagram

10b the absorption and the fluorescence spectrum of the dye CY 2 and filter characteristics of the spectral filter arrangement for maximum and minimum light incidence angles are given.

FIG. 11 specifies the spectral filter and radiation guide arrangement preferably used for the detection of the dye FITC in a representation corresponding to FIG. 10.

12 specifies those preferred for the detection of the dye

Rhodamine 6G used spectral filter and radiation guide arrangement in a representation corresponding to FIG. 10.

13 specifies those preferred for the detection of the dye

TexasRed ™ used spectral filter and Radiation guidance arrangement in a representation corresponding to FIG. 10.

FIG. 14 specifies the spectral filter and radiation guide arrangement preferably used for the detection of the dye CY 5 in a representation corresponding to FIG. 10.

15 specifies those preferred for the detection of the dye CY

5.5 USED SPECTRAL FILLING AND RADIATION RADIATION ARRANGEMENT IN A REFERENCE TO FIG. 10.

16 specifies those preferred for the detection of the dye IRD

800 used spectral filter and radiation guiding arrangement in a representation corresponding to FIG. 10.

FIG. 17 shows a diagram which summarizes some of the data from FIGS. 10 to 16 and the spectral position of the absorption and emission spectra corresponding to a relative amplitude of at least 0.2, the proposed spectral passband of the spectral filter arrangements respectively assigned to the respective fluorescent dye according to a transmission of at least 0.5 for angle of incidence a = 0 ° and, for example, possible ones

Indicates excitation wavelengths and laser types.

FIG. 18 shows a variant of the thermostatic plate of FIG. 6, with which a temperature gradient in the direction of propagation of the molecules can be set during electrophoresis. Fig. 1 9 shows in Fig. 1 9a a cross-sectional view with an electrophoresis gel layer sandwiched between two thermostatic plates and in Fig. 1 9b schematically shows a temperature gradient in the gel adjustable by the arrangement of Fig. 1 9a in a direction assigned to the thickness of the gel layer ,

Reference is first made to Figs. 1-3. Fig. 1 shows schematically a gel electrophoresis device 1 0 with a vertically arranged gel layer or gel plate 1 2, which is arranged between a so-called notched plate or notch plate 1 4 and a thermostatic plate or thermal plate 1 6. In the production of the gel layer 1 2, a row of recesses or pockets open at the top and arranged at an upper edge of the gel layer 1 2 was formed, each of which lies at the beginning of an associated gel electrophoresis path and receives the samples to be analyzed. On a gel width of 305 mm, for example, 1 92 gel pockets 1 8 and thus 1 92 gel electrophoresis sheets lying next to one another can be provided, corresponding, for example, to a sheet width of 1 mm and a sheet gap of 0.6 mm, so that, for example have 48 clones (4 lanes per clone) analyzed in a sequencing analysis per selectively detectable fluorescent marker. With regard to the manner in which the electrophoresis gel of the device according to the invention is loaded with samples to be analyzed, there are basically no restrictions; reference can be made, for example, to the techniques and methods known from WO96 / 27787 and WO98 / 3091 1.

During the electrophoresis operation, an electrical potential is applied between an upper buffer tank and a lower buffer tank 24 by means of a voltage source 20 in accordance with the charge of molecules to be analyzed. In the case of the analysis of sequencing products, the upper buffer tank 22 is opposite to the lower buffer tank is more negative potential, so that the negatively charged DNA fragments move along the electric field lines from top to bottom through the gel, usually a poly-acrylamide gel, from top to bottom (molecular propagation direction M). In the course of their movement along the respective gel electrophoresis path, the molecules, possibly DNA segments, are separated according to their molecular weight or their size or length. The migration rate of the molecules sings disproportionately with increasing molecular size or molecular weight, so that molecules of smaller molecular weight (shorter DNA fragments) move faster than molecules of larger molecular weight (longer DNA fragments) and first reach a detection area located in a lower region of the gel , Here, the molecules marked with fluorescent dyes are optically detected via fluorescence radiation emanating from the respective fluorescent marker, which is induced by optical excitation of the respective fluorescent marker by laser radiation. Due to the dependence of the molecular propagation speed (migration speed) on the molecular weight (the molecule size, possibly DNA fragment length), the molecules of smaller molecular weight (the smaller molecules or the shorter DNA fragments) first pass through the laser beam used for excitation, so that the spatial separation of the Molecules in the vertical direction (whose vertical separation distance) is converted into temporal information, namely in the order in which the molecules pass through the laser beam. In the case of the analysis of DNA sequencing products, this temporal information can be converted into sequence information, for example by means of a computer unit 26.

The thermostatic plate 1 6 is assigned a thermostattable heat exchange medium reservoir, preferably a thermostattable water bath 28, a heat exchanger circuit, if necessary, via a pump 30. Water cycle, through which the thermostat is carried out 1 6, evenly Adjust or maintain temperature conditions, especially in the horizontal direction, across all gel electrophoresis lanes. Apart from the mentioned number of electrophoresis lanes and the lane widths and interstices, the gel electrophoresis device 10, as far as described so far, corresponds to conventional electrophoresis devices. For the basic structure of such electrophoresis equipment and the background of the analysis of sequencing products by means of such a device, reference is expressly made to W096 / 2321 3. The thermostatic plate 1 6 can be designed in accordance with the thermostatic plates known from DE 30 46 729 C2. Reference is expressly made to DE 30 46 729 C2, and the disclosure of this document is incorporated by reference into the disclosure of the present description.

Further details of an electrophoresis device according to the invention in accordance with the exemplary embodiment shown in FIG. 1 are now explained below, which lead to significant advantages over the prior art.

The electrophoresis device 10 of FIG. 1 is designed to use laser radiation from five different lasers for the detection of the fluorescent markers of the marked molecules migrating along the direction M, of which only four lasers are shown in FIG. 1, namely one at 488 nm emitting argon ion laser 50, a helium-neon laser 52 emitting at 594 nm, a semiconductor laser emitting in the region of 675 nm and tunable, for example, by means of a Peltier element to the emission wavelength of 675 nm (also referred to as laser diode) 54 and an im Range of 775 nm emitting semiconductor lasers 56, for example tunable to a wavelength of 775 nm by means of a Peltier element. Of the lasers, only the actual laser or laser head having the laser resonator is shown, but not associated control devices and the like. The argon ion Laser 50 and helium-neon laser 52 are arranged with a resonator axis defining the laser beam propagation direction at least approximately horizontally in a respective horizontal plane, and at least approximately parallel to gel layer 1 2. The semiconductor lasers 54 and 56 are likewise at least approximately horizontal with their corresponding resonator axis arranged in a respective horizontal plane, at least approximately orthogonally to the gel layer 12. The horizontal planes assigned to the four lasers are arranged in a vertical staggered manner, preferably with a vertical distance of approximately 10 mm corresponding to a vertical distance of the laser beams 60, 62 emitted by the lasers , 64 and 66. The laser beams are deflected twice by deflection mirrors 70 and 72 in the case of beams 60 and 62 from argon ion laser 50 and helium-neon laser 52 and in the case of laser beams 64 and 66 by semiconductor lasers 54 and 56 once to the at least deflected approximately 90 ° in order to couple the laser beams laterally into the gel layer 1 2 in a vertically staggered manner with the aforementioned vertical spacing of, for example, approximately 10 mm. To focus the laser beams, an optical lens 74, for example a plano-convex lens, is arranged in the beam path of the laser beams, by means of which the waist of the laser beam profile, ideally a beam profile corresponding at least approximately to a Gaussian beam profile, approximately in the center of the gel in relation is placed on the width b. For fine adjustment of the course of the laser beams in the vertical direction, the lenses 74 are preferably adjustable in height, preferably adjustable in height by motor. The deflecting mirrors 72 which deflect the laser beams in the direction of the gel are preferably adjustable in a direction at least approximately orthogonal to the gel layer 1 2, preferably adjustable by motor, in order to offset the laser beams in a respective horizontal plane in the transverse direction parallel to the gel layer and thus as precisely as possible in the Couple the middle of the gel layer (based on the thickness of the gel) into the gel. An adjustment of the laser beams by means of the adjustable Optical elements are preferably based on detection signals from the respective detector field, which are based, for example, on scattered light or background fluorescent light from the gel layer and / or adjacent glass plates. A background radiation profile is preferably recorded and then a laser beam profile corresponding to a maximum and / or a center of the background radiation profile is set, advantageously in each case with respect to at least one detector field section at mutually opposite ends of the detector field. The coupling into the gel is preferably carried out via a common coupling plate which laterally delimits the gel.

It should also be added to the semiconductor lasers that their comparatively strongly divergent laser beams are bundled by means of a respective telescope, not shown in FIG. 1, in order to bring the beam diameter to less than 1 mm. The laser beams can additionally pass through a spatial filter, for example a pinhole, in order to achieve the ideal Gaussian beam. In the case of the argon-ion laser and the helium-neon laser, the laser beam profile generally corresponds quite well to a Gaussian beam profile, so that such beam conditioning is generally not necessary.

The optical components mentioned (deflection mirrors 70 and 72, lenses 74 etc.) and the semiconductor lasers 54 and 56 are mounted on a step-shaped optical bench 80, the steps 82 of which rise laterally in accordance with the offset between the laser beams, as can be seen in FIG. 1. By means of the stepped optical bench, a compact arrangement with high stability can be achieved in a particularly simple manner while enabling the laser beam paths mentioned. The top step of the stepped optical bench 82 is unused according to FIG. 1, here a further semiconductor laser with corresponding optical components or a further arrangement of deflecting mirrors 70 and 72 for a further, Lasers arranged parallel to the lasers 50 and 52 can be mounted in order to couple five laser beams in a vertically stepped manner into the gel layer.

The laser beams with the four different wavelengths serve to excite assigned fluorescent markers, for example the fluorescent markers FITC, TexasRed ™, CY 5.5 and IRD 800, which have only slightly overlapping excitation and emission spectra and thus enable selective excitation and detection (see Fig . 8th). A detection arrangement for detecting the emission radiation emitted by the excited fluorescence markers is assigned to the fluorescent dyes used and thus to the laser beams. The detector arrangements are integrated in a detector module 90 which, in the view in FIG. 1, is arranged behind the notch plate 14 and is explained in more detail with reference to FIGS. 2 and 4.

FIG. 2 shows the detector module 90 in a top view of input sides of five optics and detector field modules 94, 96, 98 and 100, of which the modules 92 to 98 are assigned to the laser beams 60, 62, 64 and 66, with FIG. 2, the ideal course of the laser beams with respect to the input sides of the optics and detector field modules is indicated in dashed lines, namely parallel to a longitudinal axis of the modules in the middle of the respective input side.

A possible construction of the optics and detector field modules is shown in FIG. 3. The optics and detector field module shown there is formed by a linear detector field 102, which has, for example, a series of photodiodes 104, and a spectral filter and radiation guide arrangement which, in the example of FIG. 3, a spectral filter 106 and a gradient index lens field, for example sucked in. SELFOC lens array (SLA) 1 08 comprises, for example, six rows of gradient index lenses vertically one above the other. The gradient index lens field 108 is preferably a lens field with a numerical aperture of approximately 0.6, corresponding to a maximum angle of incidence of a = ± 22.5 °, that is to say an opening angle of an incoming beam of rays of approximately 45 °, the individual lenses being cylindrical Elements with a diameter of 1 mm and a length of 10 mm are formed in the 6 lines mentioned, each with 300 lenses. A lens field contains approximately 1,800 such lenses and there is a total light entry area of 305 mm × 6 mm. Such lens fields are available as custom-made products from the Nippon Sheet Glass Company, Japan.

The linear detector field 1 02 preferably comprises highly sensitive photodiodes with a dimension of the light-sensitive surfaces of about 3 mm, with a view to differentiating the gel electrophoresis sheets at least two photodiodes (pixels) per electrophoresis sheet should be provided, so that the linear Detector field preferably has at least 384 photodiodes arranged side by side. The linear detector array 104 is preferably formed from six modules lined up in a linear manner, which can be implemented in hybrid construction and can each carry 64 photodiodes on a common chip with a width of 2 inches (5.08 cm), preferably together with associated integrating operational amplifiers. Good results were achieved with a light-sensitive total area of 30.5 cm × 3 mm per detector line, divided into the 384 photodiodes mentioned, each 0.8 mm wide. To minimize the light-insensitive area, the light-insensitive zones between the individual photodiodes should be as narrow as possible. In the preferred embodiment, the light-insensitive zone between the photodiodes is only 0.02 mm each, so that the entire light-insensitive area 383 x 0.2 mm is equal to 7.66 mm, corresponding to a very small proportion of only 2.5% of the total detector surface. Such photodiode modules are custom-made available from Hamamatsu Photonics, Japan. The spectral sensitivity of the silicon photodiodes is shown in FIG. 7 and certain limits can be set by changing the doping.

The gradient index lenses of the gradient index lens field 108 each generate mutually overlapping individual images on the image side, that is to say on the photodiodes, with a spatially continuous 1-to-1 imaging occurring. Because of this and because of the high numerical aperture on the one hand and the dimensions of the photodiodes in the Miekü propagation direction M (3 mm in the example described here) on the other hand, the orientation of the gradient index lens field 108 with respect to the detector field 108 is not particularly demanding.

Instead of a linear detector field formed from photodiodes, a CCD field or another spatially resolving detector field can also be used, which enables a spatially resolved detection of the fluorescent light along a detection path which is defined by the course of the laser beams in the gel. The detector fields used can certainly have a matrix of detection pixels with several rows of pixels parallel to one another. The term “linear” used in relation to the detector fields is not restrictive in this respect and relates to the possibility of spatially resolved fluorescence light detection along a linear detection path according to the exemplary embodiment.

Absorption filters (for example colored glass filters) and interference filters and, above all, combinations of at least one absorption filter and at least one interference filter come into consideration as spectral filters. The passband of the spectral filter of the various optics and detector field modules is to be selected in accordance with the excitation wavelengths and the emission spectra of the fluorescent dyes used, in the following for a number of usable fluorescent markers suitable spectral filter specifications are given as an example. Remarkably, fluorescent markers with overlapping emission spectra can also be distinguished from one another, provided that these can be selectively excited and spatially separated laser beam excitation takes place, for example by means of mutually offset laser beams as in the exemplary embodiment in FIGS. 1 and 2. Alternatively, temporally separated laser beam excitation and excitation with differently modulated laser beams can also be considered to enable phase-sensitive detection. Furthermore, it is also possible to differentiate selectively or non-selectively excitable fluorescent markers with overlapping emission spectra, provided that the emission spectra only overlap in regions, i.e. do not have overlapping spectral ranges and the emission radiation is detected in a spectrally selective manner, possibly in connection with spatially separated laser beam excitation or / and a temporally separated laser beam excitation or / and an excitation with modulated laser radiation to enable phase-sensitive detection.

A particularly preferred embodiment of the spectral filter and radiation guide arrangements of the detector module, each as an integral optical module, is shown in FIGS. 4 and 5 in spatial and functional relation to a linear detector field separate from the integral optical module. To explain the embodiment variant of FIGS. 4 and 5, the same reference numerals are used as for the embodiment of FIGS. 1 to 3 for analog or identical components followed by the letter "a", only the differences from the previously described embodiment being described and otherwise, reference is expressly made to the preceding description.

FIG. 4 schematically shows the arrangement of the optical and electronic elements of a detector unit assigned to a laser beam with the Detector field (of the detector line) in a sectional view with the direction of view corresponding to the direction of molecular propagation M (in the arrangement according to FIG. 1, therefore, from above) and FIG. 5 shows the elements of the detector unit in a sectional plane orthogonal to the gel layer, the notch plate and the thermostat plate, whereby the photodetectors of the detector line in Fig. 4 are only indicated schematically.

According to the variant of FIGS. 4 and 5, an integral optical module for the respective laser is provided for guiding the fluorescence marker emission radiation resulting from the excitation by an excitation laser, which consists of a gradient index lens field 1 08a, an absorption filter 106a on the image side of the Gradient index lens array 1 08a, an optical interference filter 1 07a on the object side of the gradient index lens array 108a and a mirrored deflection prism made of high-index flint glass SF 4 1 10a. The spectral filters 106a, 1 07a and the deflection prism 1 10a are glued directly onto the gradient index lens field 108a and onto an aluminum carrier rail in order to obtain an optical unit with a defined optical path length. The beam path for the emission radiation from the excited fluorescent markers can be seen in FIG. 5. The optical path length Z (Λ) is approximately 20 mm, the optical path length required for the imaging being achieved on the basis of a relatively short geometric path length due to the highly refractive deflection prism. In order to match the optical path length to the different fluorescence wavelengths, deflection prisms with different refractive indexes can be used for the individual optical modules assigned to the detector rows. To a certain extent, however, different path lengths caused by chromatic aberration can also be compensated for by the fact that the optical units 1 1 2a can be adjusted by shifting on the optical axis. To form the respective optical unit 1 05a from the filters 1 06a, 1 07a, the gradient index lens field 108a and the deflecting prism 1 10a can also be added that the bonding can be carried out, for example, by means of an optical adhesive based on silicone, which preferably has a low viscosity and is preferably adapted in terms of its refractive index to the refractive indices of the filter glasses and the gradient index lens field.

Like the detector field 102, the linear detector field 102a assigned to the respective fluorescent dye consists of six modules arranged in a linear manner, which are plugged in on a common carrier board 1 1 4a, on which clock generators and multiplexers 1 1 6a are also accommodated. The individual modules are designed in hybrid construction and each carry 64 photodiodes on a common chip with a width of two inches (= 5.08 cm), together with the associated integrating operational amplifiers. The entire light-sensitive area of a detector line is thus 30.5 cm wide and 3 mm high, divided into 385 photodiodes 0.8 mm wide. The light-insensitive zone between the individual photodiodes is 0.02 mm wide. The entire light-insensitive area is accordingly 383 x 0.02 mm = 7.66 mm wide, corresponding to a very small proportion of only 2.5% of the total detector area.

The gradient index lens field 108a can correspond to the gradient index lens field 108 and, for example, have a high numerical aperture NA of approximately 0.6 and six rows of lens elements one above the other in order to ensure a large vertical detection range for fluorescence radiation (object height) of 3 mm with a full numerical aperture. Due to the overlapping upright image of the individual lenses in the field, a horizontal alignment of the lens elements with the photodiodes is not absolutely necessary. The carrier board 1 14a with the detector fields 102a arranged thereon and the optical modules 1 1 2a are received and held in a common detector module according to FIG. 2 with a common, shielding housing. 1, the detector module 90 can thus have five carrier boards 114a with a respective detector field 102a and five associated integral optical modules 11 2a. Due to the bent beam path achieved by the deflection prisms 1 10a, the minimum vertical distance between the individual detection arrangements is determined by the structural height of the gradient index lens fields 108a, which is approximately 10 mm. When using approximately 60 cm long glass plates to limit the gel layer, separation distances (running distance of the molecules or fragments to be analyzed) from 46 cm to the top detector or from 50 cm to the bottom detector can be achieved.

The linear detector fields can be formed by silicon photodiodes, which have a wide spectral sensitivity range (see FIG. 7). However, other materials, for example gallium arsenide phosphite, can also be used. The latter semiconductor material has the great advantage that the spectral sensitivity can be shifted into a wide range by varying the proportions of arsenide and phosphite in order to provide a special adaptation to the emission radiation to be detected in each case. Furthermore, this material is relatively insensitive to thermal background radiation compared to silicon. One could also think of further increasing the pixel density of the detector fields in order to achieve a higher track density, for example a doubling or tripling of the track density.

The electrophoresis device in the form shown in FIG. 1 can be used, for example, to analyze molecules which are marked by four different fluorescent markers, for example FITC, TexasRed ™, CY 5.5 and IRD 800 (cf. FIG. 8). To prevent premature degradation of the To avoid fluorescent markers by laser radiation, which has a shorter wavelength than the laser radiation used for excitation, the lasers and the laser beams are staggered vertically in such a way that the excitation wavelengths increase along the direction of molecular propagation M, so that the fluorescence marker-labeled molecules first have the long-wave laser radiation of 775 nm, then the shorter-wave laser radiation of 675 nm, then the laser radiation of 594 nm and finally the laser radiation of the shortest wavelength of 488 nm. A corresponding arrangement is preferably also provided when other excitation wavelengths and other laser sources are used.

Basically, there are no restrictions with regard to the fluorescent dyes that can be used. An overview of fluorescent dyes that can be used for DNA sequencing with the maxima of the respective excitation and emission spectrum and suitable excitation light sources is given in Table 1.

Table 1

In connection with an electrophoresis device according to the invention according to FIG. 1, the dyes CY 2, FITC, Rhodamine 6G, TexasRed ™, CY 5, CY 5.5 and IRD 800 have proven particularly useful. For many applications it will be sufficient if the electrophoresis device is designed with regard to the design of its detectors in such a way that only four of the laser-dye combinations can be used simultaneously. For some of the dyes listed in Table 1, the chemical structure is shown in Fig. 9.

It should be noted that the excitation lasers shown in Fig. 1 serve only as an example. Table 2 gives examples of excitation lasers that can be used for DNA sequencing with their emission wavelength Λ _em and thereby fluorochromes that can be excited. The right column of the table shows the product of the extinction coefficient e (λ _em ) and the quantum yield q (Λ _em ) of the respective dye at the corresponding excitation wavelength Λ _em , which is a measure of the fluorescence yield that can be achieved. Since the excitation takes place selectively at the respective absorption maximum, only low laser output powers are required.

Table 2

An overview of the types of lasers that can be used in an electrophoresis device according to the invention with wavelengths, output powers and the respective absorption maximum of selectively excitable fluorescent dyes is given in Table 3. By selective excitation of the fluorochromes close to the respective absorption maximum, only low laser output powers between 3 and 5 mW are required. Higher laser powers can even be harmful, since effects such as photo-bleaching of the dyes, radiation-induced chemical reactions of the dyes with the gel matrix and change in their refractive index, and an increase in the background due to scattered radiation can occur. Furthermore, the yield of fluorescence does not increase in proportion to the power of the excitation light source, since saturation can occur due to the depletion of the energetic ground state of the fluorochromes. Laser types whose output power cannot be regulated or can only be regulated with difficulty can therefore be weakened, for example, by means of optical neutral filters in the beam path. Table 3

With reference to FIGS. 10 to 1 6, the spectral properties of the dyes CY 2, FITC, Rhodamine 6G, Texas Red, CY 5, CY 5.5 and IRD 800 are to be used for the detection of the respective emission radiation according to the exemplary embodiment described here Deta tto purities, especially l of the spectral filter arrangements used in connection with the properties of the respective gradient index lens field. The use of the optical radiation filter arrangement makes it possible to separate the fluorescent light from the excitation light of the lasers and partly from the background fluorescence as well as from Raylaigh and Raman scattered light in order to be able to detect the comparatively much weaker fluorescent light at all. By selective excitation and detection of the individual fluorescent markers (fluorochromes) it is further ensured that several differently colored molecules, possibly DNA fragments, can be analyzed simultaneously but independently of one another in the same electrophoresis gel. The optical radiation filters can be band-pass filters in which areas with low transmission (cut-off areas) are connected to a region of high transmission (pass band) towards shorter and longer wavelengths and / or edge filters in which a region of high transmission is followed by a region of low transmission connects, or vice versa, where long-pass filters in which the pass band is at longer wavelengths than the stop band, and / or short-pass filters in which the pass band is shorter than the stop band can be used as edge filters. Interference filters, which can be combined with absorption short-pass and / or absorption long-pass filters, are preferably used as bandpass filters. Depending on the gradient index lens field used, the possible total thickness of the filter arrangement can be set to a certain value, for example approx. 2 mm, for optimal imaging properties of the optical system, but in principle compensation is also possible by using materials with a correspondingly adapted refractive index. A particularly simple optical structure, which can also be easily adjusted, is achieved if the spectral filters have a predetermined total thickness for all detectors. The filter characteristic of the respective absorption filter can then be adapted primarily via the melt used for the production of the absorption filter. In this context, it is appropriate that a respective absorption filter is used as the substrate for the production of the interference filter. Short-pass and long-pass filters from Schott (Mainz, Germany) can be used as absorption filters.

The central data of the spectral filter arrangement used to detect the fluorescent light of the fluorescent marker CY 2 according to a preferred exemplary embodiment are given in the table in FIG. 10a. A combination of an absorption short-pass filter, an absorption long-pass filter and an interference band-pass filter is used as the filter Edge wavelengths λ _c1 , λ _C2 of 494 nm and 524 nm for a light incidence angle a = 0 ° and Λ _C1 = 492 nm and λ _C2 = 514 nm for a light incidence angle a = 20 ° corresponding to a maximum acceptance angle of the gradient index lens field. Here, an edge wavelength is denoted in each case by a wavelength giving a transmission of 0.5 (50%). In the preferably used excitation wavelength of 473 nm, the transmittance of the spectral filter is less than 1 0 ^"4 in the light incident angle range 0 ° to 20 °. Is obtained at a light incident angle of 0 ° for the wavelength range 495 for the emission spectrum of the dye to 51 5 nm, a transmission With a light incidence angle of 20 °, a transmission greater than 0.8 is obtained for the wavelength range 490 to 505 nm. For wavelengths greater than the emission spectrum of the dye, a transmission less than 10 ^{~ 3 is obtained} for the light incidence angle range 0 10b shows a diagram which indicates the transmission of the spectral filter arrangement as a function of the wavelength for a = 0 ° and for a = 20 ° In addition, the absorption spectrum A and the emission spectrum E of the dye CY 2 and that are preferred used excitation wavelength 473 nm is drawn in. This is also preferred for the excitation of the dye rhodamine 6G (cf. Fig. 1 2) used excitation wavelength of 532 nm.

Fig. 1 1 shows corresponding data for a detector assigned to the dye FITC. The spectral filter arrangement in turn preferably comprises an absorption short-pass filter, an absorption long-pass filter and an interference band-pass filter. In addition to excitation by means of an argon ion laser with a wavelength of 488 nm, the use of a frequency-doubled neodymium: YAG laser with a wavelength of 473 nm can also be considered, for example.

The data of a detector assigned to the dye rhodamine 6G are shown in FIG. 1 2. The spectral filter arrangement preferably consists of a Interference bandpass filter and an absorption shortpass filter, the transmission of which is additionally indicated in FIG. 1 2b (curve K).

The fluorescent marker TexasRed ™ can be detected, for example, by means of a detector, the spectral specifications of which are given in FIG. 1 3. The spectral filter arrangement is preferably formed by an interference bandpass filter and an absorption longpass filter. The transmission of the long-pass filter is also shown in Fig. 1 3b (curve L).

A spectral filter arrangement formed by an absorption long-pass filter and an interference band-pass filter can be used, for example, for the detection of the fluorescent light emitted by the dye CY 5, the spectral data of which are given in FIG. 14. In addition to excitation by means of a helium-neon laser with the emission wavelength of λ = 632.8 nm, excitation by means of a semiconductor laser, for example with a wavelength of 640 nm, can also be considered.

The dye CY 5.5 can be detected, for example, by means of a spectral filter arrangement which is formed by an absorption long-pass filter and an interference band-pass filter. The spectral data of a suitable detector are given in Fig. 1 5. The dye CY 5.5 can be excited, for example, by means of a laser diode which emits at 675 nm. A semiconductor laser diode emitting at 660 nm can also be used. The detector specified in FIG. 15 can also be used for the detection of the dye IRD 700.

For the detection of the dye IRD 800, which is chemically identical to the fluorochrome IRD 41, a detector can be used, its spectral

Specifications are given in Fig. 1 6. The spectral filter arrangement can consist of an absorption long-pass filter and an interference band-pass filter be educated. The dye can be excited with a laser diode that emits at 775 nm, for example. With regard to the emission spectrum (E) in Fig. 1 6b and the corresponding spectrum in Fig. '8, it should be noted that the decrease in emission shown towards longer wavelengths should be somewhat exaggerated.

The excitation and absorption spectra of the dyes mentioned, the pass bands of the assigned spectral filter arrangements to be understood as examples, and the excitation wavelengths in question to be understood as examples are summarized in the diagram in FIG. 1 7, of the absorption spectra (A) and the emission spectra (E) in each case the range of a relative signal strength standardized to 1> 0.2 and for the spectral filters in each case the pass range for the light incidence angle σ = 0 ° is given. As can be seen, there are spectrally wide overlaps between the absorption spectra of the dyes and between the emission spectra of the dyes. Despite these overlaps, the use of four independent detection systems enables four samples labeled with different fluorescent dyes to be detected simultaneously and independently of one another, for example samples labeled with the dyes fluorescein, TexasRed, CY 5.5 and IRD 800 (four different dyes). Furthermore, these five dyes can be detected simultaneously and independently of one another by means of the detector units specified for the five dyes CY 2, Rhodamine 6G, TexasRed, CY 5.5 and IRD 800. On the basis of the teaching of the present invention, the person skilled in the art will easily be able to set up an electrophoresis device with five or more independent detection systems which enables simultaneous detection independently of one another for another dye combination of five dyes or for more than five assigned different fluorescent dyes , The crosstalk of the four detectors for the dyes fluorescein, TexasRed, CY 5.5 and IRD 800 was determined quantitatively as follows: After measuring the thermal background in complete darkness, i.e. lasers switched off, the laser beams assigned to the four detectors were coupled into an electrophoresis gel onto which no samples were applied. The lasers were switched on one after the other with a time interval of about 15 minutes. The measured values for the background radiation were in each case averaged over 40 photodiodes. The following table 4 shows the rise in the output voltage of the detector in digital units of the A / D converter used in absolute terms and as a percentage (based on the purely thermal radiation background). The increase caused by the laser assigned to the respective detector is highlighted in bold in the table.

Table 4

Further tests showed that the increase in the radiation background (maximum 34% for the detector for CY 5.5) is constant over time and does not change during the course of the electrophoresis operation. This increase can thus be subtracted arithmetically as a constant background from the electrophoresis useful signal, so that the influence of the background radiation increased by the laser radiation on the signal-to-noise ratio of the respective detector and thus on the separability of the system can be largely neglected.

In order to investigate possible radiation of the fluorescence emission light of the dyes into the detectors not assigned to them, a sequencing run was carried out for the four dyes with samples which were labeled with only this one of the four dyes. Labeled primers were used. During the sequencing run, the signals from the non-corresponding detectors were also recorded. The strongest signals appeared at the beginning of each sequencing run as the primers passed through the laser beams. In all cases, only this passage through the primers could be recognized in the other detectors, i n c ht a d a r th th e lo c ting of the sequencing products. So there was no detectable interference in the area of the actual sequence signals. The spectral selectivity of the detector system was therefore sufficient to analyze the DNA samples labeled with different dyes simultaneously, but independently of one another.

Table 5 and Table 6 show results on the resolving power of the detection system with regard to the distances between the electrophoresis tracks (tracks) and the separation distance. Table 5

Table 6

Table 5 shows that the achievable resolving power is largely independent of the distance between the tracks on the electrophoresis gel, as long as the Nyquist scanning theorem is adhered to, which requires at least two pixels (in the present exemplary embodiment formed by photodiodes) per track. Table 6 makes it clear that doubling the separation distance from 21 cm to 42 cm by using 60 cm long thermostated glass plates brings a considerable advantage, namely an increase in the average number of bases read by 40%, so that with 60 cm long gels in one Run over 1,200 bases was read. To However, it should be taken into account that the beam diameter in the electrophoresis gel is not constant in the beam propagation direction. Ideally, the beam waist of the laser beam is placed in the middle of the gel so that the highest resolution can be expected here. The resolution then decreases towards the edges in accordance with the beam diameter and the divergence angle of the laser beam. On average, reading lengths of over 800 bases were achieved.

The following are examples of the analysis of nucleic acids using an electrophoresis device according to the invention:

example 1

1 .1 Material and methods

Primer design and synthesis

Either standard primers (UPO, UP-40, RPO, T7, T7term) or the specific primers 1 to 8 specified below were used as primers. Suitable vectors with inserts to be sequenced were used as matrices for the respective primers. The fluorescent dyes fluorescein isothiocyanate (FITC), TexasRed- (TR), CY 5.5 and IRD 800 were used as labels.

The sequences and labels of the primers used are summarized in Table 7. Table 7

Sequenzierprotokolle

All sequencing reactions were performed as thermocycling reactions (Murray et al., Nucleic Acids Res. 17 (1 989), 8889) using a thermal cycler (MJ Research, PTC-200) with the following profile: 25 or 40 cycles at 97 ° C for 1 5 sec, 55 ° C for 30 sec, 68 ° C for 60 sec or 30 sec.

The protocols for the reactions (number of primers - number of templates - number of reaction vessels) were as follows:

Tandem duplex response (4-2-1)

3 μl of DNA template 1 (1 μg / μl) were mixed with 2 μ \ DNA template 2 (0.5 μg / μl) and 3 μ each of the primers FITC-T7term (4 μM), TR-T7, CY 5.5-RPO and IRD 800-UPO (2 μM each), 2, 1 μ \ 10x cycling buffer, 3 μ \ AmpliTaqFS (Applied Biosystems) and 1.9 ul water mixed. The mixture was divided into four aliquots (5 μ \ each). 3 μ \ of the respective termination mixture (A, C, G, T) were added to the individual aliquots. Then thermal cycling was started. After 25 cycles, 1μ stop solution was added to each reaction and the volume reduced by about half during denaturation at 95 ° C for about 10 minutes. Then the batch was put on hold.

Quadruplex reaction (4-4-1) 2 μ \ DNA template 1 (0.5 μg / μl) was combined with 3 μ \ DNA template 2 (0.3 μg / μl), 5 μ \ DNA template 3 (0, 2 μg / μl), 3 μ \ DNA template 4 (0.3 μg / μl) and 1 μ each of primers 1 to 4 (5 μM each), 2.4 μl 1 X X cycling buffer, 3 μl AmpliTaqFS and 1 , 5 ul DMSO mixed. The mixture was divided into four aliquots (5 μl each) and 3 μl of the respective termination mixture (A, C, G, T) were pipetted into the individual aliquots. Then thermal cycling was started. After 25 cycles, 1 ul stop solution was added to each reaction and the volume reduced by about half during denaturation at 95 ° C for about 10 minutes. Then the approaches were put on hold.

Sequence gap filling reaction (4-1 -1)

1.5 μl DNA template 1 (0.7 μg / μl) were mixed with 2 μl each of primers 5 to 8 (2 μM each), 2.1 μl 10 X cycling buffer, 3 μl AmpliTaqFS, 1.5 μl DMSO and 4 , 9 ul water mixed. The mixture was divided into four aliquots (5 μl each) and 3 μl of the respective termination mixture (A, C, G, T) were pipetted into the individual aliquots. Then thermal cycling was started. After 25 cycles, 1 µl of stop solution was added to each reaction and the volume was reduced by about half during a denaturation at 95 ° C for about 10 minutes. Then the approaches were put on hold.

Competitive tandem duplex reaction (4-1-1) 4 μl DNA template 1 (1 μg / μl) were each with 2 μl primers (FITC-RP, TR-40, RPO-CY 5.5, IRD 800-40, each 2 μM), 2.0 μl 10 X cycling buffer, 3 μl AmpliTaqFS and 3 μl water mixed. The mixture was divided into four aliquots (5 μl each) and 3 μl of the respective termination mixture (A, C, G, T) were pipetted into the individual aliquots. Then thermal cycling was started. After 40 cycles (extension at 68 ° C for 60 sec), 1 ul stop solution was added to each reaction batch and the volume was reduced to about half during a denaturation at 95 ° C for about 10 min. Then the approaches were put on hold.

Corresponding results were also achieved with the reagents of the commercial cycle sequencing kit from Applied Biosystems (TaqFS Dye Primer Cycle Sequencing Kit).

1 .2 Automated DNA sequencer

To excite the four fluorescent dyes, four lasers with different emission wavelengths were used, which had been selected in accordance with the respective absorption spectrum of the fluorescent dye. The laser types and powers are shown in Table 8 below.

Table 8

The lasers were coupled into the gel via a single coupling plate at four different positions with a vertical distance of 10 mm between the beams. The beam position was fine-tuned individually for all four lasers using a motorized optical stage.

According to the emission spectra of the four fluorescent dyes, optical bandpass filters were selected for the optimal transmission of the respective emission light spectra and the maximum suppression of scattered excitation laser light and of the infrared background due to glass and gel fluorescence. The high selectivity of the transmission profile was achieved by a combination of absorption and multilayer interference filters (Schott, Mainz, Germany).

The light was collected and focused on the detectors by an optical system that provides a spatially continuous and upright image, high resolution and a high aperture (i.e. minimal photon loss). A gradient index lens array (Nippon Sheet Glass Company, Japan) was used to collect and image fluorescent light.

The fluorescent light was detected by means of four stacked detector arrays (Hamamatsu Photonics, Japan), onto which the fluorescent light was imaged by a respective gradient index lens array (cf. the explanations above for the exemplary embodiment of the electrophoresis device).

1 .3 Gel electrophoresis

The products of the sequencing reactions were separated on 5.0% or 4.3% Hydrolink Long Ranger denaturing gels (AT Biochem, Malvern, PA, USA) with 42% urea. Running and gel buffers were 1, 2 x TBE. 100 ml of gel solution were polymerized by adding 84.5 μl of N, N, N ′, N′-tetramethylethylenediamine (TEMED) and 650 μl of 10% ammonium peroxodisulfate (APS) solution in water. The gels were 0.30 mm thick. The temperature was 45 ° C. The separation was carried out over a distance of approximately 50 cm using 60 cm long and 34 cm wide glass plates. The electrophoresis was carried out with a power of 60 W and an initial voltage of about 1500 V.

The sequences of all four differently labeled reaction products with a single gel web were detected on-line and recorded in a computer. The evaluation was carried out automatically after the end of the run using the software packages Lanetracker and Geneskipper (EMBL, Schwager et al.).

1.4 Gel loading

To handle the large number of samples and the simultaneous loading of the 120 traces of a gel, combs made of porous filter material and a robot system (Beckman Biomek 2000) were used. This method is described in detail by Erfle et al. (Nucleic Acids Res. 25 (1997), 2229-2230) and includes loading the wells of a polyacrylic (Plexiglas) block with the sample suspension by a pipetting robot. By dipping the teeth of the comb into the pockets of the plexiglass block, the porous material absorbs the sample liquid by capillary action. The comb containing the samples is then inserted between the glass plates just above the straight edge of the polymerized gel. When the electric field is applied, the samples are pulled out of the comb and into the gel.

0.6 μl of the sequencing samples were loaded onto the comb using this technique. In addition, four standard sequencing reactions (4-4-4) and two duplex Sequencing reactions (4-2-2) carried out in separate reaction vessels, premixed and loaded onto the comb or loaded separately onto the same teeth of the comb without premixing all four reactions. The results obtained were identical to those of the protocols described above.

1.5 results

The fluorescence quadruplex sequencing method allows the use of different strategies. Four different matrices were thus prepared in one reaction vessel using four differently labeled primers (4-4-1), two different matrices with a tandem duplex sequence reaction in two directions and four differently labeled primers in one vessel (4-2-1). and sequence gap filling reactions were successfully carried out using four "walking primers" to simultaneously close four gaps on the same template in a vessel (4-1-1).

With a resolution of 1000 bases per strand, about 4000 bases per sequencing reaction could be determined by the techniques described. This means that 1 20 sequencing reactions can be separated on a single gel, thereby obtaining 1 20 kb information per gel.

The excitation of the fluorescent dyes and the collection of emission light took place continuously over the width of the gel and continuously over the entire duration. Each dye could be selectively excited and detected. The accuracy and readable sequence length showed no difference compared to standard sequencing reactions. The fluorescence quadruplex sequencing method which can be carried out by means of the electrophoresis device according to the invention is also suitable for internal labels or dye-labeled terminators.

Example 2

Using the protocol described in Example 1, 1,92 sequencing reactions (i.e. 48 sequencing reactions on independent clones in each case) were separated simultaneously on a single gel. Thereby, 1 55 kb or more information per gel was obtained.

Example 3

The dye FITC used in Example 1 was replaced by 5-carboxy-rhodamine 6G. Instead of the argon laser, a neodymium-YAG laser with an emission wavelength of 532 nm and a power of 5 to 10 mW was used. Here too, selective excitation and selective detection of four dyes on a gel sheet was possible.

Example 4

In addition to the dye combination described in Example 3, dye CY 2 was used. A neodymium YAG laser with a wavelength of 473 nm and a power of 5 to 10 mW was used as the excitation light source.

It has been found that selective excitation and selective detection is possible even when five fluorescent dyes are used simultaneously on a gel sheet.

The invention is explained in more detail below with regard to further aspects on the basis of exemplary embodiments. To temper the gel, an electrophoresis device according to the invention can have a thermostattable plate of the type shown in FIG. 6 as an example. FIG. 6 corresponds to FIG. 1 of DE 3046 729 C2 already referred to. The thermostattable plate 1 6b consists of a lower glass plate 1 1 2 and an upper glass plate 21 4, which are arranged in parallel one above the other and are held at a defined distance from one another by a multi-part spacer strip 21 6. The two glass plates 21 2 and 21 4 preferably have the same outline, but can have different thicknesses, for example 3 mm for the lower glass plate 21 2 and 1.5 mm for the upper glass plate 214 which is in contact with the gel layer. The outer upper side of the upper glass plate 214 can be pretreated in order to be gel-repellent and thus to facilitate the detachment of the gel layer coming into contact with the upper side. The two glass plates, like the multi-part spacer strip 21 6, are made of mirror glass or floated glass in order to ensure high planicity.

The spacer strip 21 6 runs along the rectangular outline of the glass plates 21 2 and 214. It consists of two six sections running from top to bottom in FIG. 1, the left one being designated 220 and the right one being 222, and two of them Cross sections 224 and 226 connecting ends.

The spacer strip 21 6 is self-contained and therefore includes an interior 228 between the glass plates 21 2 and 21 4. To through the interior 21 8 heat exchanger means, for. B. via a separate thermostat heating water or from the thermal bath and pump assembly 28, 30 (Fig. 1) water, two connections are provided, an inlet connection 230 and an outlet connection 232. The inlet connections are by means of the Glue used glue tightly integrated into the spacer strips 21 6. A plurality of guide strips 240 are used in the interior 228 in order to achieve a meandering (zigzag-shaped or serpentine) flow path 238 for the heat exchange medium. Between the guide strips 240, the flow direction C of the heat exchanger means preferably perpendicular to the direction of molecular propagation M, which in the illustration in FIG. 6 deviates from the illustration in FIG. 1 from bottom to top. The thermostattable plate can be arranged for use in electrophoresis for vertical separation from top to bottom as desired. The conductive strips 240 can be made of glass or - alternatively - made of a good heat-conducting material, e.g. B. copper exist. The latter can improve the temperature constancy in the transverse direction to the molecular propagation direction M.

A modified embodiment of the thermostattable plate is shown in Fig. 1 8. The thermostattable plate 16c shown here has continuous guide strips 240 'in the transverse direction, which divide the interior 228' into seven separate flow areas 239 '. In addition to the connections 230 'and 232', further connections 234 'are provided, so that each flow area 239' has an inlet connection and an outlet connection. Connections immediately following one another along the direction of molecular propagation M can be connected to one another in pairs in order to achieve a meandering passage of the heat exchanger means through the plate, as in the embodiment of FIG. 6. Alternatively, each flow area 239 'can be supplied with heat exchange medium with a differently set temperature in order to set a temperature gradient in the thermostating plate 16c along the molecular propagation direction M. For example, a temperature profile T, indicated schematically on the right of the plate 1 6c, can be set, according to which the temperature of the plate and thus of the adjacent gel layer increases in the direction of molecular propagation M, while in the transverse direction a substantially constant temperature prevails. It can be a temperature profile that increases linearly or also parabolically. In the case of 60 cm long plates, a temperature rise over a few degrees, for example 2 to 10 °, along the plate in the molecular propagation direction has proven to be advantageous in order to achieve a kind of focusing effect for the bands moving in the molecular propagation direction M by influencing the viscosity of the gel , This focusing effect is based on the front flanks of the bands moving from a higher mobility area to a lower mobility area. This focusing compensates, at least to a certain extent, for the band diffusion that naturally occurs in electrophoresis, and higher resolutions and longer sequence reading lengths can be achieved.

The thermostattable plate enables the setting of a wide variety of temperature profiles, for example also a temperature profile with increasing temperature in the direction of molecular propagation. Furthermore, it may be advantageous to provide a locally elevated temperature in an initial region of the gel electrophoresis tracks in order to create more denaturing conditions for the DNA.

The detection of the bands by means of at least one laser beam can be supported in that in the area of the laser beam cut by the laser beam. If a temperature rise, that is a locally higher temperature, is set. This leads to a targeted broadening of the bands and an increase in the band spacing, which results in a better detectability of the bands.

It has been found that a focusing temperature gradient in the molecular propagation direction can also be achieved in other ways using a thermostatted plate as shown in Fig. 6, based on a special mode of operation for the thermostatted plate. After this mode of operation, the vertical in a thermostatted plate arranged in a housing is first heated to a predetermined temperature, for example 45 ° C., by means of the heat exchanger medium circuit in order to create denaturing conditions in the gel and to heat the gel uniformly in order to ensure the conditions for a uniform and parallel migration of the mole cules to be analyzed in g el to create. The heat exchanger medium circuit is then interrupted by the thermostattable plate, for example by uncoupling the plate from the water bath. In this way, on the one hand it is achieved that vibrations of the thermostat plate induced by pressure fluctuations of the heat exchanger means are eliminated, which lead to non-planarities of the thermostat plate and thus via a modification of the laser beam guidance in the gel, in particular additional reflections of the respective laser beam on the glass of the thermostat plate, an increase in the background - Radiation background (additional scattered light and / or additional fluorescent light due to an inherent fluorescence of the glass) causes, whereby the signal-to-noise ratio is deteriorated and thus the sequence reading length is limited. It also arises due to heat exchange with the surroundings of the thermostattable plate in the surrounding housing of the electrophoresis device (not shown in FIG. 1), especially the ambient atmosphere, and possibly due to a convection. The surrounding atmosphere or / and the heat exchange medium in the plate enter a temperature gradient in the direction of molecular propagation M, with a higher temperature at the upper end of the gel, i.e. at the beginning of the gel electrophoresis webs, and a lower temperature at the lower End of the gel, i.e. at the end of the gel electrophoresis lanes. Despite the interruption of the heat exchange medium circuit, the general temperature level, apart from the temperature gradient, is maintained at least to a certain extent due to the Joule heat developed in the gel during electrophoresis, so that the denaturing conditions in the gel are maintained. This way too using a thermostattable plate as shown in FIG. 6, a temperature profile falling by a few degrees in the direction of molecular propagation can be achieved in order to achieve the focusing effect explained.

By arranging the thermostatic plate appropriately and, if necessary, additional shielding measures, other temperature profiles, possibly with a local temperature increase in at least one transverse area of the gel, can be achieved.

A kind of focusing effect can also be obtained on the basis of a temperature profile with a temperature gradient in a direction corresponding to a thickness of the gel layer. On the basis of the Joule heat generated in the gel during electrophoresis and, for example, two thermostattable plates 1 6d 1 and 1 6d2, between which the gel layer 1 2d is arranged (see FIG. 1 9a), a temperature gradient can be set accordingly in the direction mentioned a temperature profile at which the temperature in the middle of the gel is at a maximum and decreases towards the thermostattable plates. A corresponding temperature profile is shown as an example in Fig. 1 9b. Since the migrating molecules concentrate in an area of higher mobility, i.e. lower viscosity, there is an accumulation of molecules of the same molecular weight (same fragment length) in a central area of the gel layer, which reduces disruptive effects at the interface between adjacent glass plates and the gel , This also has a focusing effect on the migrating bands. A temperature gradient can be provided both in the direction of molecular propagation M and in the direction associated with the gel thickness. Alternatively, a temperature gradient can only be provided in the direction of molecular propagation M or only in the direction associated with the gel thickness. The invention relates to an electrophoresis device for analyzing fluorescent marker-labeled molecules, in particular biological molecules, which according to one aspect of the invention is suitable for independently of one another at least four different fluorescent markers by means of an associated detector field according to the respective gel electrophoresis pathway and the to detect the respective fluorescent marker in a resolved manner. For this purpose, spectral filters are matched to one another in a corresponding manner with regard to their spectral selectivity, the fluorescent markers with regard to their spectral absorption and emission selectivity and the wavelengths of the laser beams used to excite the fluorescent markers.

Claims

Expectations

Electrophoresis device (1 0) for analyzing fluorescence marker-labeled molecules, in particular biological

Molecules comprising: a plurality of gel electrophoresis tracks (1 2), which run essentially parallel to one another and along a molecular propagation direction (M), - an energy source (20) for applying an electrical one

Potentials at an initial region and an end region of the gel electrophoresis sheets (1 2), a laser arrangement (50, 52, 54, 56) for exciting a plurality of different fluorescent markers assigned to the molecules, at least some of which have absorption wavelength ranges which differ from one another (A) and / or emission wavelength ranges (E) different from one another, the laser arrangement in electrophoresis mode generating laser radiation which is considered to be substantially transverse to that

Molecular propagation direction and laser beams (60, 62, 64, 66) which run essentially parallel to one another and are mutually offset in the molecular propagation direction cuts at least several of the gel electrophoresis tracks (1 2) in order to detect the respective gel area in a detection area.

To stimulate fluorescence markers associated with the electrophoresis pathway of the molecules present there, a detection arrangement (90) for gel electrophoresis pathway-resolved and fluorescence marker-resolved detection of those emanating from the excited fluorescence markers

Emission radiation wherein the detection arrangement has a plurality of detector fields (102; 102a) which are offset from one another in the direction of molecular propagation and preferably extend essentially parallel to the laser beams, each of which is assigned to a specific one of the laser beams and is designed for this purpose, preferably along a path assigned to the respective laser beam to detect at least a part of this essentially parallel detection path in a spatially resolved manner in the detection areas which arise in the detection areas due to the excitation by the respective laser beam and which are guided to the detector field via a respectively assigned spectral filter and radiation guide arrangement (106, 108; 112a), the spectral filter arrangement (106; 106a, 107) with regard to their spectral selectivity, the fluorescent markers with regard to their spectral absorption and emission selectivity, and the wavelengths of the laser beams are matched to one another in such a way that using at least four laser beams and at least four

Detector fields at least four different fluorescence markers can be detected independently of one another by means of an assigned detector field according to the respective gel electrophoresis path and the respective fluorescence marker.

2. Electrophoresis device (10) for analyzing fluorescence marker-labeled molecules, in particular biological molecules, comprising: a plurality of gel electrophoresis tracks (12) which run essentially parallel to one another and along their direction of molecular propagation (M), an energy source (20) for applying an electrical potential to a start region and an end region of the gel electrophoresis tracks (1 2), a laser arrangement (50, 52, 54, 56) for exciting a plurality of different molecules assigned to them

Fluorescence markers, at least some of which have absorption wavelength ranges (A) that are different from one another and / or emission wavelength ranges (E) that differ from one another, the laser arrangement in electrophoresis mode

Generates laser radiation which, as laser beams (60, 62, 64, 66) which run essentially transversely to the molecular propagation direction, cuts at least several of the gel electrophoresis tracks (1 2) in order to detect the respective gel area in a detection area pho rese- Ba bn zu geo rd n ete

To excite fluorescent markers of the molecules present there, at least some of the laser beams (60, 62, 64, 66) being spatially offset from one another or / and occurring in offset time windows or / and wherein at least one laser beam is modulated to enable phase-sensitive detection and / or wherein at least some of the laser beams have different wavelengths from one another, a detection arrangement (90) for gel electrophoresis path-resolved and fluorescence marker-resolved detection of emission radiation emitted by the excited fluorescence markers, wherein the detection arrangement extends at least one preferably essentially parallel to the laser beams Has detector field (102; 1 02a), which is designed to be along an associated, preferably substantially parallel to the laser beam Detection path to detect at least part of the emission radiation which is generated in the detection areas due to the excitation by the respective laser beam and is spatially resolved via an assigned spectral filter and radiation guide arrangement (1 06, 1 08; 1 1 2a), the spectral filter arrangement (106; 106a , 1 07) with regard to their spectral selectivity, the fluorescence markers with regard to their spectral absorption and emission selectivity, and the wavelengths of the laser beams are matched to one another in such a way that using at least four laser beams, at least four different fluorescence markers independently of one another according to the respective gel Electrophoresis web and the respective fluorescent marker are resolvably detectable.

3. Device according to claim 1 or 2, characterized in that the laser radiation selectively excitable fluorescent markers with overlapping emission spectra (E) can be distinguished from one another on the basis of spatially and / or temporally separate laser beam excitation and / and on the basis of different wavelengths of the laser beams (60, 62 , 64, 66) or / and on the basis of a phase-sensitive detection upon excitation with modulated laser radiation and / or that fluorescence markers which can be selectively or non-selectively excited by the laser radiation with at least partially non-overlapping emission spectra (E) can be distinguished on the basis of spectrally selective detection of the emission radiation and possibly spatially separated laser beam excitation.

4. Device according to one of claims 1 to 3, characterized in that the wavelengths of the laser beams (60, 62, 64, 66) and / or spectral filter (1 06; 1 06a, 1 07a) of Spectral filter arrangements are matched to at least four different fluorescence markers whose absorption maxima are at least 30 nm apart, preferably at least 50 nm apart, under electrophoresis operating conditions, and / or whose emission maxima are separated under electrophoresis.

Operating conditions are at least 30 nm apart, preferably at least 50 nm apart.

5. Device according to one of claims 1 to 4, characterized in that at least one spectral filter (106; 106a, 107a) has a spectral characteristic such that due to the excitation of the respectively assigned laser beam, fluorescent radiation of the respectively assigned fluorescent marker for a pass wavelength range is created around an emission maximum of the fluorescence spectrum of this fluorescence marker or for a transmission wavelength range adjacent to this fluorescence maximum is guided to the assigned detector field (102; 102a).

6. Device according to claim 5, characterized in that in relation to at least one of the other fluorescent markers at least one of the following conditions a) and b) is fulfilled in order to enable the detection resolved after the fluorescent markers: a) the laser beam wavelength is present one spectral side of an absorption spectrum (A) of the other

Fluorescence marker beyond an edge of the absorption spectrum falling from an absorption maximum to the laser beam wavelength down to at least a residual absorption amplitude, which corresponds to an at most low residual absorption, b) the transmission wavelength range lies on one spectral side of an emission spectrum (E) of the other F luo resz n zma rke rs on the one hand an emission maximum to the pass wavelength range down to at least one residual emission amplitude, which corresponds to an at most low residual emission, falling edge of the emission spectrum.

7. Device according to claim 6, characterized in that the falling edge to the laser beam wavelength is a falling edge towards longer wavelengths and the falling edge to the pass wavelength range is a shorter edge

Is the falling edge.

8. Device according to claim 6, characterized in that the flank falling to the laser beam wavelength is a flank falling to shorter wavelengths flank and the pass-through

Wavelength falling flank is a flank falling towards longer wavelengths.

9. Device according to one of claims 6 to 8, characterized in that the flank to the laser beam wavelength or to

The transmission wavelength range drops more steeply than an edge of the spectrum falling on the other side of the maximum of the spectrum (A or E) and falling with the distance from the maximum.

10. Device according to one of claims 1 to 9, characterized in that at least one spectral filter and radiation guide arrangement as a radiation guide arrangement comprises a lens arrangement, preferably a gradient index lens field (1 08; 1 08a).

1 1. Device according to one of claims 1 to 1 0, characterized by at least one on one of the gel electrophoresis tracks assigned object-side spectral filter (107a) or / and at least one image-side spectral filter (106; 106a) arranged on an image side of the radiation-guiding arrangement assigned to the detector field.

12. Device according to one of claims 1 to 11, characterized in that at least one spectral filter arrangement (106a, 107a) is designed as an interference filter or comprises an interference filter (107a).

13. Device according to one of claims 1 to 12, characterized in that at least one spectral filter arrangement (106a, 107a) is designed as a combination of an absorption filter arrangement (106a) and an interference filter arrangement (107a).

14. The device according to claim 13, characterized in that at least one spectral filter (107a) comprises an absorption filter, possibly colored glass filter, which serves as a carrier substrate for thin-film films of an associated interference filter.

15. Device according to one of claims 12 to 14, characterized in that the interference filter (107a), possibly in conjunction with the radiation guide arrangement (108a), defines a wavelength range as a transmission wavelength range, which one

Superimposition of passband of an / the interference filter for different light incidence angles in the interference filter corresponds.

16. ' Apparatus according to claim 15, characterized in that a wavelength range is defined as the transmission wavelength range, which risti kder all transmission ranges of the interference filter (107a) for all acc Radiation guide arrangement (108a) occurring light incidence angle.

1 7. A device according to claim 1 5 or 1 6, characterized in that a wavelength range is defined as the transmission wavelength range, which is a transmission range of the interference filter (107 a) for a light incidence angle of a = 0 ° and a transmission range of the interference filter for one the maximum acceptance angle of the beam guiding sensor (1 08 a) includes the corresponding angle of incidence.

18. Device according to one of claims 1 to 1 7, characterized in that at least seven markers can be detected from the following fluorescent markers: indodicarbocyanine CY 2, Alexa 488, fluorescein iso-thio-cyanate (FITC), 5-carboxy-

Rhodamine 6G, Alexa 532, Tetramethyl-Rhodamine-Iso-Thio-Cyanate (TRITC), Indodicarbocyanin CY 3, Sulforhodamine 101 (TexasRed ™), Indodicarbocyanin CY 5, Indodicarbocyanin CY 5.5, IRD 700, Indodicarbocyanin CY 7, IRD 40 and IRD ,

1 9. Device according to claim 1 8, characterized in that at least the markers indodicarbocyanine CY 2, fluorescein-iso-thio-cyanate (FITC), 5-carboxy-rhodamine 6G, sulforhodamine 1 01 (TexasRed ™), indodicarbocyanine CY 5, Indodicarbocyanin CY 5.5 and IRD 800 are detectable.

20. Device according to one of claims 1 to 1 9, characterized in that at least three, preferably at least four, most preferably at least five markers of the following fluorescent markers are simultaneously dissolved and dissolved

G el- Electrophoresis- Bah n- can be detected in dissolved form: indodicarbocyanine CY 2, Alexa 488, fluorescein isothio cyanate (FITC), 5-carboxy-rhodamine 6G, Alexa 532, tetramethyl-rhodamine-iso-thio-cyanate (TRITC), indodicarbocyanine CY 3, sulforhodamine 101 (TexasRed ™), indodicarbocyanine CY 5, indodicarbocyanine CY 5.5, IRD 700, indodicarbocyanine CY 7, IRD 40 and IRD 800.

21. Device according to claim 20, characterized in that at least the five markers

Indodicarbocyanine CY 2, 5-carboxy-rhodamine 6G,

Sulforhodamine 101 (TexasRed ™), Indodicarbocyanin CY 5.5 and IRD 800 or at least the four markers

Fluorescein iso-thio-cyanate (FITC), sulforhodamine 1 01 (TexasRed ™), indodicarbocyanine CY 5.5 and IRD 800 - or at least the four markers indodicarbocyanine CY 2, 5-carboxy-rhodamine 6G,

Indodicarbocyanin CY 5 and IRD 800 or at least the three markers

Fluorescein iso-thio-cyanate (FITC), indodicarbocyanine CY 5 and IRD 800 are simultaneously fluorescent marker-resolved and Ge!

22. Device according to one of claims 1 8 to 21, characterized in that a frequency-doubled, possibly diode-pumped neodymium: YAG laser with an emission wavelength of approximately 473 nm is provided for the detection of the fluorescent marker indodicarbocyanine CY 2.

23. Device according to one of claims 1 8 to 22, characterized in that the fluorescent marker indodicarbocyanine CY

2 shows a spectral filter arrangement with a pass band of approximately 494 nm to approximately 524 nm, possibly with different light Passage areas associated with angles of incidence a of approximately 494 nm to approximately 524 nm for a = 0 ° and approximately 492 nm to approximately 514 nm for a light angle of incidence corresponding to a maximum angle of the radiation guiding arrangement, for example of approximately 20 °, are assigned.

24. Device according to one of claims 18 to 23, characterized in that one / the spectral filter arrangement assigned to the fluorescent marker indodicarbocyanine CY 2 has an absorption short-pass filter, an absorption long-pass filter and one

Interference bandpass filter includes.

25. Device according to one of claims 18 to 24, characterized in that for the detection of the fluorescent marker fluorescein iso-thio-cyanate (FITC) an argon ion laser with a

Emission wavelength of approximately 488 nm or a frequency-doubled, possibly diode-pumped neodymium: YAG laser with an emission wavelength of approximately 473 nm is provided.

26. Device according to one of claims 18 to 25, characterized in that the fluorescence marker fluorescein-iso-thio-cyanate (FITC) is assigned a spectral filter arrangement with a pass range from about 505 nm to about 555 nm, possibly with different light incidence angles a Pass ranges from approximately 505 nm to approximately 555 nm for a = 0 ° and approximately 500 nm to approximately 545 nm for a maximum angle of incidence corresponding to the radiation guiding arrangement corresponding to the light incidence angle, for example of approximately 20 °, are assigned.

27. Device according to one of claims 18 to 26, characterized in that one / the spectral filter arrangement associated with the fluorescein iso-thio-cyanate (FITC) Absorption short pass filter, an absorption long pass filter and an interference band pass filter.

28. Device according to one of claims 18 to 27, characterized in that for the detection of the fluorescent marker 5-

Carboxy-Rhodamine 6G a frequency-doubled, possibly diode-pumped neodymium: YAG laser with an emission wavelength of about 532 nm is provided.

29. Device according to one of claims 18 to 28, characterized in that the fluorescence marker 5-carboxγ-rhodamine 6G has a spectral filter arrangement with a pass band of about 555 nm to about 578 nm, possibly with different pass angles associated with different pass angles a of about 555 nm to about 578 nm for a = 0 ° and about 553 nm to about 569 nm for a maximum angle between the radiation guiding arrangement corresponding light incidence angle, for example of about 20 °, is assigned.

30. Device according to one of claims 18 to 29, characterized in that one / the spectral filter arrangement assigned to the fluorescent marker 5-carboxy-rhodamine 6G comprises an absorption short-pass filter and an interference band-pass filter.

31 .. Device according to one of claims 18 to 30, characterized in that a helium-neon laser with an emission wavelength of about 594 nm is provided for the detection of the fluorescent marker sulforhodamine 101 (TexasRed ™).

32. Device according to one of claims 18 to 31, characterized in that the fluorescent marker sulforhodamine 101 (TexasRed ™) has a spectral filter arrangement with a passband from about 613 nm to about 651 nm, possibly with transmission ranges assigned from different angles of incidence a of about 613 nm to about 651 nm for a = 0 ° and about 613 nm to about 636 nm for a maximum acceptance wi n ke I the radiation guide arrangement is assigned corresponding light incidence angles, for example of approximately 20 °.

33. Device according to one of claims 18 to 32, characterized in that one / the one assigned to the fluorescent marker Sulforhodamine 101 (TexasRed ™) spectral filter arrangement

Absorption long pass filter and an interference band pass filter.

34. Device according to one of claims 18 to 33, characterized in that a helium-neon laser with a for the detection of the fluorescent marker indodicarbocyanine CY 5

Emission wavelength of about 633 nm or a semiconductor laser with an emission wavelength of about 640 nm is provided.

35. Device according to one of claims 18 to 34, characterized in that the fluorescent marker indodicarbocyanine CY

5 shows a spectral filter arrangement with a pass band from approximately 655 nm to approximately 690 nm, possibly with pass band ranges from approximately 655 nm to approximately 690 nm associated with different light incidence angles a for a = 0 ° and approximately 650 nm to approximately 683 nm for a maximum Acceptance angle of the

The radiation guide arrangement is assigned corresponding light incidence angles, for example of approximately 20 °.

36. Device according to one of claims 18 to 35, characterized in that a / the fluorescent marker

Spectral filter arrangement assigned to indodicarbocyanine CY 5 comprises an absorption long-pass filter and an interference band-pass filter.

37. Device according to one of claims 1 8 to 36, characterized in that a semiconductor laser with an emission wavelength of about 660 nm or about 670 nm or about 675 nm is provided for the detection of the fluorescent marker indodicarbocyanine CY 5.5 or / and the fluorescent marker IRD 700 ,

38. Device according to one of claims 1 8 to 37, characterized in that the fluorescent marker indodicarbocyanine CY 5.5 or / and the fluorescent marker IRD 700 a spectral filter arrangement with a pass range from about 655 nm to about 690 nm, possibly with different light incidence angles a assigned transmission ranges from approximately 695 nm to approximately 745 nm for a = 0 ° and approximately 695 nm to approximately 730 nm for a light incidence angle corresponding to a maximum acceptance angle of the radiation guide arrangement, for example of approximately 20 °.

39. Device according to one of claims 1 8 to 38, characterized in that a / which the fluorescence marker indodicarbocyanine CY 5.5 or / and the fluorescence marker IRD 700 assigned spectral filter arrangement comprises an absorption long-pass filter and an interference band-pass filter.

40. Device according to one of claims 18 to 39, characterized in that for the detection of the fluorescent marker IRD

800, a semiconductor laser with an emission wavelength of approximately 775 nm or of approximately 780 nm is provided.

41. Device according to one of claims 1 8 to 40, characterized in that the fluorescent marker IRD 800 a

Spectral filter arrangement with a pass band from approximately 795 nm to approximately 790 nm, possibly with different light incidence angles a assigned transmission ranges from approximately 795 nm to approximately 835 nm for a = 0 ° and approximately 790 nm to approximately 830 nm for a light incidence angle corresponding to a maximum acceptance angle of the radiation guide arrangement, for example of approximately 20 °.

42. Device according to one of claims 18 to 41, characterized in that a / the spectral filter arrangement assigned to the fluorescent marker IRD 800 comprises an absorption long-pass filter and an interference band-pass filter.

43. Device according to one of claims 1 to 42, characterized by measures for reducing the resolution of the detection in relation to the gel electrophoresis web and / or in relation to the fluorescent markers and / or the signal-to-

Noise ratio interfering interference radiation, possibly fluorescent light background radiation and / or scattered laser radiation.

44. Device according to claim 43, characterized in that at least one of the laser beams is linearly polarized with a polarization vector which is after an input side

Light guiding direction of the spectral filter and radiation guide arrangement is aligned, possibly at least approximately parallel to this.

45. Device according to claim 43 or 44, characterized in that the gel electrophoresis tracks run in a preferably plate-shaped gel matrix (12) which is arranged between glass plates (14, 16) made of floated borosilicate glass.

46. Device according to one of claims 43 to 45, characterized in that the gel electrophoresis tracks run in a preferably plate-shaped gel matrix (1 2), the laser beams (60, 62, 64, 66) via at least one to the laser beam - Direction of propagation in the gel matrix (1 2) essentially parallel

Coupling plate are coupled into the gel matrix, the coupling plate having a polished entry surface that is essentially orthogonal to the respective laser beam and / or a polished exit surface or / and an orthogonal to the respective laser beam that is adapted to the gel matrix, the refractive index of the

Gel matrix optionally has at least approximately corresponding refractive index.

47. Device according to claim 46, characterized in that a common coupling plate is provided for all laser beams (60, 62, 64, 66).

48. Device according to one of claims 1 to 47, characterized in that the spatially offset, different wavelengths having laser beams (60, 62,

64, 66) are offset from one another in such a way that the laser radiation wavelength decreases in the molecular propagation direction (M) from laser beam to laser beam.

49. Electrophoresis device (1 0) for analyzing fluorescence marker-labeled molecules, in particular biological molecules, comprising: a plurality of gel electrophoresis tracks (12) which run essentially parallel to one another and along their direction of molecular propagation (M), an energy source (20) for applying an electrical potential to a start region and an end region of the gel electrophoresis tracks (12), a laser arrangement (50, 52, 54, 56) for exciting fluorescence markers assigned to the molecules, the laser arrangement being in electrophoresis mode Generates laser radiation which, as at least one laser beam (60, 62, 64, 66) running essentially transversely to the molecular propagation direction, cuts at least several of the gel electrophoresis tracks in order to be in one

To excite the detection area of the respective gel electrophoresis path assigned fluorescence markers of the molecules present there, a detection arrangement (90) for detecting those emanating from the excited fluorescence markers

Emission radiation, wherein the detection arrangement has at least one detector field (102; 102a) which preferably extends essentially parallel to the at least one laser beam and which is designed to along at least a part of the detection paths associated with the laser beam, preferably essentially parallel to the latter Detection areas resulting from the laser beam excitation via an assigned spectral filter and radiation guide arrangement (106, 108; 112a) for

Detect field-guided emission radiation to be recorded in a spatially resolved manner, the spectral filter and radiation guide arrangement emitting the emission radiation emanating from the excited fluorescence markers on receiving surfaces or

Receiving surface portions (104a) of the detector array (102a) deflected, which are arranged at least approximately orthogonally to the direction of molecular propagation (M).

50. Device according to claim 49, characterized in that the detector field (102a) is arranged on a carrier board (114a) which is arranged at least approximately orthogonally to the direction of molecular propagation (M).

51. Device according to claim 49 or 50, characterized in that several one behind the other in the molecular propagation direction

A plurality of detector beams (102a), preferably assigned to different wavelengths, are provided, each with receiving surfaces or receiving surface sections (104a) that are at least approximately orthogonal to the direction of molecular propagation (M), which are preferably each arranged on a separate carrier board (114a).

52. Device according to one of claims 49 to 51, characterized in that the spectral filter and

Radiation guidance arrangement (112a) comprises at least one deflection prism arrangement which is assigned to a respective detector field (102a) and extends essentially parallel to this detector field, possibly a continuous deflection prism (110a).

53. Device according to claim 52, characterized in that the deflecting prism arrangement (110a) is arranged on an output side of a lens arrangement, possibly a gradient index lens field (108a), of the spectral filter and radiation guide arrangement.

54. Device according to one of claims 49 to 53, characterized in that the deflecting prism arrangement (1 10a) is formed from a highly refractive optical material for adapting an image-side optical path between a / the lens arrangement, possibly a / the gradient index lens field (1 08a) , the spectral filter and radiation guiding arrangement on the one hand and the receiving surfaces (104a) on the other hand to an image-side focal length of the lens arrangement.

55. Device according to one of claims 52 to 54, characterized in that the deflecting prism arrangement (1 1 0a) is part of an integral optical module (1 1 2a) which has a / the lens arrangement (1 08A) and at least one spectral filter (106a, 1 07a).

56. Device according to claim 55, characterized in that the deflecting prism arrangement (1 10a), the lens arrangement (108a) designed as a gradient index lens field, the at least one spectral filter (106a, 107a) and, if desired, a carrier rail for producing the integral optical module (1 1 2a) are glued together.

57. Device according to claim 56, characterized in that on an optical input side of the gradient index lens field (1 08a) and / or on an optical output side of the gradient index field

(1 08a), optionally between the gradient index lens field and the deflecting prism arrangement (1 10a), at least one spectral filter (1 06a) is glued on.

58. Device according to claim 56 or 57, characterized in that at least one spectral filter (107a) of the optical module (1 1 2a) is designed as an interference filter or comprises an interference filter.

59. Device according to one of claims 56 to 58, characterized in that at least one spectral filter (107a) is designed as a combination of an absorption filter and an interference filter.

60. Device according to claim 59, characterized in that at least one spectral filter (107a) of the optical module comprises an absorption filter, possibly colored glass filter, which serves as a carrier substrate for thin-film films of an associated interference filter.

61. Device according to one of Claims 56 to 60, characterized in that the optical module (1 1 2a) for adjusting an optical path in a module carrier in a laser beam section which intersects the gel electrophoresis tracks (1 2) and the direction of molecular propagation in the essential orthogonal

Direction is arranged.

62. Device according to one of claims 55 to 61, characterized in that a plurality of detector fields (102a), a plurality of optical modules (1 1 2a) each assigned to one of the detector fields and detection electronics (1 1 6a) assigned to the detector fields in one Detector module (90) are integrated with a common housing, the detector module being able to be handled as a unit and preferably by being moved in a direction that cuts the gel electrophoresis paths

Laser beam sections and to the molecular propagation direction (M) essentially orthogonal direction allows a common adjustment of assigned object-side optical path lengths for all optical modules (1 1 2a) of the detector module.

63. Device according to one of claims 49 to 62, characterized by the remaining features of at least one of the preceding claims 1 to 48.

64. Electrophoresis device (1 0) for analyzing fluorescence marker-labeled molecules, in particular biological molecules, comprising: a plurality of gel electrophoresis tracks (1 2) which run essentially parallel to one another and along a molecular propagation direction (M), an energy source (20) for applying an electrical

Potentials at an initial region and an end region of the

Gel electrophoresis sheets, a laser assembly (50, 52, 54, 56) for exciting a plurality of different ones associated with the molecules

Fluorescence markers, wherein the laser arrangement in the electrophoresis mode generates laser radiation, which as at least several of the gel electrophoresis tracks (60, 62, 64, 66) of different wavelengths, which run essentially transversely to the molecular propagation direction and essentially parallel to one another and offset in the molecular propagation direction with respect to one another. 1 2) cuts a detection arrangement (90) for gel electrophoresis path-resolved and fluorescence marker-resolved detection of emission radiation emanating from the excited fluorescence markers, in order to excite fluorescence markers of the molecules present in a detection region assigned to the respective gel electrophoresis pathway, whereby the Detection arrangement a plurality of mutually offset and in the molecular propagation direction preferably essentially parallel to the

Has laser fields extending detector fields (102; 10a), which are each assigned to a specific one of the laser beams and are designed for this purpose, along at least one detection path assigned to the respective laser beam, preferably essentially parallel to the latter

Part of the radiation guidance arrangement (106, 108; 1 1 2a) that arises in the detection areas due to the excitation by the respective laser beam, via a newly-designed device

Detection of the emission radiation guided by the detector field, the laser arrangement having at least one laser (50, 52) with an optical laser resonator, which is at least approximately parallel to the detector fields in relation to a resonator axis that characterizes the beam path of the laser beam (60 or 62) emerging from the resonator (102; 1 02a) is arranged, the laser beam (60 or 62) emerging from the laser resonator being deflected by means of a beam guiding arrangement (70, 72), so that a beam which runs transversely to the molecular propagation direction

Laser beam section cuts the gel electrophoresis sheets (1 2).

65. Device according to claim 64, characterized in that a plurality of lasers (50, 52) with at least approximately parallel resonators with respect to a respective resonator axis are provided with the detector fields, the laser resonators with respect to the resonator axes at least approximately corresponding to the offset of the gel Electrophoresis tracks (1 2) intersecting laser beam sections in the molecular propagation direction (M) are offset from each other.

6. electrophoresis device (10), optionally according to claim 64 or 65, for analyzing fluorescence marker-labeled molecules, in particular biological molecules, comprising: a plurality of substantially parallel to one another and along a molecular propagation direction (M)

Electrophoresis sheets (1 2), an energy source (20) for applying an electrical potential to an initial region and an end region of the gel electrophoresis sheets (1 2), - a laser arrangement (50, 52, 54, 56) for exciting one

A plurality of different fluorescent markers assigned to the molecules, the laser arrangement in electrophoresis mode generating laser radiation which has at least several wavelengths as different laser beams (60, 62, 64, 66) which are essentially transverse to the direction of molecular propagation and essentially parallel to one another and offset in the direction of molecular propagation the gel electrophoresis lanes (1 2), in order to excite fluorescence markers of the molecules present in a detection area associated with the respective gel electrophoresis lane, cuts a detection arrangement (90) for gel electrophoresis lane-resolved and fluorescence marker-resolved detection of them excited fluorescent markers

Emission radiation, the detection arrangement having a plurality of mutually offset in the molecular propagation direction and preferably extending essentially parallel to the laser beams detector fields (102; 102a), each associated with a specific one of the laser beams and designed for this, along one Associated with the respective laser beam, preferably at least substantially parallel to this detection path, at least some of the spectra I fi - ter - and - generated in the detection areas due to the excitation by the respective laser beam

Radiation guidance arrangement (106, 108; 1 1 2a) to detect emission radiation guided to the detector field in a spatially resolved manner, the laser arrangement having at least two lasers (54, 56) with a respective laser resonator, each with reference to one

The beam path of the laser beam (64 or 66) which characterizes the respective resonator is arranged at an angle to the detector fields (1 02; 1 02a), preferably at least approximately orthogonally to the detector fields (102; 102a), the axis being out The laser beam (64 or 66) emerging from the respective laser resonator is deflected by means of a beam guiding arrangement (72), so that a laser beam section running transversely to the molecular propagation direction cuts the gel electrophoresis paths (1 2) and the laser resonators at least approximately correspond to the resonator axes the offset of the laser beam sections intersecting the gel electrophoresis tracks in the molecular propagation direction (M) are offset from one another and have different lateral spacing from the gel electrophoresis tracks (1 2).

67. Device according to claim 66, characterized in that the resonator axes lie at least approximately in a respective plane orthogonal to the direction of molecular propagation (M).

68. Device according to one of claims 64 to 67, characterized by a side of the gel electrophoresis sheets (1 2) arranged stepped optical bench (80) with steps (82) arranged at an angle to the molecular propagation direction (M), preferably at least approximately orthogonally to the molecular propagation direction (M), which with increasing lateral distance from the gel electrophoresis webs

(1 2) counter to the direction of molecular propagation (M), at least approximately in accordance with the offset of the laser beam sections cutting the gel electrophoresis tracks (12), and are associated with a respective one of the lasers for holding a respective one assigned to the laser

Beam guidance arrangement (70, 72 or 72) and / or the laser resonator (54, 56) which extends at least approximately along the step with its resonator axis.

69. Device according to claim 68, characterized in that the respective beam guiding arrangement is an optical fiber, preferably single-mode fiber, and / or at least one deflecting mirror (72) and / or at least one optical element for focusing the laser beam and / or adjusting a laser beam waist in relation to the gel electrophoresis tracks, optionally an optical lens (74), an optical objective or an optical telescope, and / or an optical element for conditioning, possibly spatial filtering, the laser beam.

70. Device according to claim 69, characterized in that at least one deflecting mirror (72) and / or the optical element (74) for adjusting the beam path of the respective laser beam is adjustable, preferably adjustable by motor.

71. Device according to Claim 70, characterized in that at least one deflecting mirror (72), from which the laser beam section cutting the gel electrophoresis tracks originates can be moved approximately in a direction orthogonal to the direction of molecular propagation (M) and the laser beam section, preferably at least approximately along the respective step (82) of the optical bench (80), for adjusting the laser beam section cutting the gel electrophoresis tracks (12).

72. Device according to claim 70 or 71, characterized in that at least one optical element (74) is at least approximately displaceable in the molecular propagation direction (M) for adjusting the cutting of the gel electrophoresis webs (12)

Laser beam section.

73. Electrophoresis device (10), optionally according to one of claims 64 to 72, for analyzing molecules marked with fluorescent markers, in particular biological molecules, comprising: a plurality of gel electrophoresis running essentially parallel to one another and along a direction of molecular propagation (M) -Bahn (12), - an energy source (20) for applying an electrical

Potential at an initial region and an end region of the gel electrophoresis tracks (12), a laser arrangement (50, 52, 54, 56) for exciting a plurality of fluorescence markers assigned to the molecules, the laser arrangement in the electrophoresis mode generating laser radiation which is used as at least one laser beam (60, 62, 64, 66) substantially transverse to the molecular propagation direction cuts at least several of the gel electrophoresis sheets (12) to be in one

Detection area of the respective gel electrophoresis web to excite assigned fluorescent markers of the molecules present there, a detection arrangement (90) for gel electrophoresis path-resolved and fluorescence marker-resolved detection of those emanating from the excited fluorescence markers

Emission radiation, wherein the detection arrangement has at least one detector field (102; 102a) which preferably extends essentially parallel to the laser beams and which is designed to along at least a portion of the in the detection areas along a detection path assigned to the laser beam, preferably essentially parallel to this the excitation caused by the assigned laser beam, via an assigned spectral filter and radiation guide arrangement

(1 06, 1 08; 1 1 2a) to detect emission radiation guided to the detector field in a spatially resolved manner, the laser arrangement having at least one laser, the laser beam of which is coupled into an optical fiber, preferably single-mode fiber, in order to direct the laser beam to the gel electrophoresis

To guide paths and / or to condition the laser beam, or / and wherein the laser arrangement has at least one laser, the laser beam of which is guided through at least one optical element for conditioning, possibly spatial filtering, the laser beam.

Device according to one of Claims 69 to 73, characterized in that the laser beam which may have a non-Gaussian profile beforehand is conditioned in such a way that it is at least approximately a Gaussian profile or / and a reduced divergence or / and a reduced beam waist -Diameter or / and one reduced, with the product of a divergence angle describing the divergence angle and the beam waist diameter increasing collimation factor, possibly so-called M ² factor.

75. Device according to one of claims 64 to 74, characterized in that at least one of the laser beams (60, 62, 64, 66) is guided and possibly focused in such a way that a laser beam region of minimal laser beam diameter, possibly one / the beam waist, is along of the assigned detector field (102; 102a) is arranged approximately in the area of a central one of the gel - electrophoresis tracks (1 2) cut by the laser beam.

76. Device according to one of claims 64 to 75, characterized by the remaining features of at least one of the preceding

Claims 1 to 63.

77. Adjustment method for adjusting the course of at least one laser beam (60, 62, 64, 66) with respect to associated gel electrophoresis tracks of an electrophoresis device, the at least one excitation laser for generating the laser beam and at least one detector field (102; 102a) for recording emission radiation emitted by excited fluorescent markers, characterized in that the adjustment is based on a detector field (1 02;

102a) recorded detection signals is carried out, which is caused by the laser signal and the signal frequency, frequency, frequency, frequency and frequency. Background fluorescence and / or scattered light.

78. Adjustment method according to claim 77, characterized in that, in the course of the adjustment, at least one optical element (72, 74) assigned to the laser beam, possibly a lens (74), a lens Telescope or a deflecting mirror (72) is adjusted to accommodate a background radiation profile, and the optical element is then set such that a background radiation is subsequently detected at least approximately according to a maximum and / or a center of the profile.

79. Adjustment method according to claim 77 or 78, characterized in that first a laser beam course intersecting the gel electrophoresis tracks (1 2) and then an at least approximately orthogonal to the gel electrophoresis tracks (1 2)

Laser beam path or / and a laser beam path that is at least approximately parallel to the assigned detector field (102; 102a) is set.

80. Adjustment method according to one of claims 77 to 79, characterized in that a laser beam path cutting the gel electrophoresis tracks is based primarily on through at least one detector field section at one end, preferably at that of a laser beam coupling point in / the gel electrophoresis tracks having the gel matrix next end, the detector field (102; 102a) recorded detection signals and a laser beam path at least approximately orthogonal to the gel electrophoresis tracks or / and a laser beam path at least approximately parallel to the detector field primarily based on at least one detector field section at the other end of the detector field recorded detection signals is set.

81. Adjustment method according to one of claims 77 to 80, characterized in that the adjustment is carried out by motor and preferably automatically.

82. Adjustment method according to one of claims 77 to 81, which is applied to a device (10) according to one of claims 1 to 76.

83. An electrophoresis device (10) for analyzing labeled molecules, in particular biological molecules, comprising: a plurality of gel electrophoresis tracks (12) which run essentially parallel to one another and along a molecular propagation direction (M) and which are arranged in a gel matrix (12 ), possibly a plate gel (12), at least one thermostatic plate (16; 16a; 16c; 16d) which is in heat exchange connection with a gel matrix surface, an energy source (20) for applying an electrical potential to an initial region and an end region of the

Gel electrophoresis sheets (12), wherein the thermostatic plate (16; 16a; 16c; 16d) is suitable for a uniform temperature distribution in the gel matrix in the

To produce and / or maintain gel electrophoresis traversing the transverse direction to the molecular propagation direction, wherein the at least one thermostatic plate (16c) is also suitable in the gel matrix (12) for a temperature gradient in the molecular propagation direction (M) or / and in a thickness of the gel matrix (12) assigned to the thermostatic plate (16d) essentially orthogonal direction, and / or is suitable for setting a temperature profile with a local temperature increase or a local temperature decrease in at least one transverse region of the gel matrix, in the case of a thermostat plate through which a heat exchanger means flows (16c) this is suitable for the relevant temperature gradient or the temperature profile in the Set the state of continuous flow through the thermostat plate (1 6c) with the heat exchanger agent.

84. Device according to claim 83, characterized in that two thermostatic plates (16d1, 1 6d2) are provided, between which the gel matrix (1 2d) is arranged.

85. Device according to claim 83 or 84, characterized in that a temperature profile (T) with decreasing or increasing temperature in the molecular propagation direction (M) can be set.

86. Device according to one of claims 83 to 85, characterized in that, based on the direction assigned to the thickness of the gel matrix (1 2d), a temperature profile (T) can be set with a temperature maximum lying in a central region of the gel matrix (1 2d).

87. Device according to one of claims 83 to 86, characterized in that a local temperature increase in a transverse region of the gel matrix corresponding to an initial region of the gel electrophoresis webs can be set.

88. Device according to one of claims 83 to 87, characterized in that a laser arrangement (50, 52, 54, 56) for exciting a plurality of the molecules assigned

Fluorescence markers are provided, the laser arrangement in the electrophoresis mode generating laser radiation which, as at least one laser beam (60, 62, 64, 66) which runs essentially transversely to the molecular propagation direction, cuts at least several of the gel electrophoresis tracks (1 2) in order to Detection range of the respective gel To excite fluorescence markers of the molecules present there associated with the electrophoresis path, wherein a local temperature increase can be set in a transverse region of the gel matrix enclosing the respective detection region.

89. Device according to one of claims 83 to 88, characterized by the remaining features of at least one of the preceding

Claims 1 to 76.

90. A method for operating an electrophoresis device (10) for analyzing labeled molecules, in particular biological molecules, which comprises: a plurality of gels which run essentially parallel to one another and along a direction of molecular propagation (M).

Electrophoresis sheets (1 2), which run in a gel matrix (1 2), possibly a plate gel (1 2), at least one thermostatic plate that is in heat exchange connection with a gel matrix surface and through which a heat exchanger means can flow

(1 6b), which is suitable for producing and / or maintaining a uniform temperature distribution in the gel matrix (1 2) in the cross direction cutting the gel electrophoresis tracks in the transverse direction to the molecular propagation direction (M), an energy source (20) for applying an electrical potential at an initial region and an end region of the gel electrophoresis sheets (1 2), characterized by the step of stopping the flow of the heat exchanger medium through the through the

Heat exchanger means preheated thermostat plate (1 6b) after reaching a predefined / predeterminable gel temperature or / and a predefined / predefinable gel temperature distribution.

91. A method according to claim 90, characterized in that after interruption of the flow in the gel matrix (1 2)

Temperature gradient in the direction of molecular propagation (M) or / and in a direction assigned to a thickness of the gel matrix, which is essentially orthogonal to the thermostatic plate (1 6b), and / or that a temperature profile with a local temperature increase or a local temperature decrease occurs in at least one transverse region of the gel matrix ,

92. The method according to claim 90 or 91, characterized in that two thermostatic plates (1 6d1, 1 6d2) are provided, between which the gel matrix (1 2d) is arranged.

93. The method according to claim 89 or 90, characterized in that a temperature profile (T) is set with decreasing or increasing temperature in the molecular propagation direction (M).

94. The method according to any one of claims 91 to 93, characterized in that, based on the direction assigned to the thickness of the gel matrix (1 2d), a temperature profile (T) is set with a temperature maximum lying in a central region of the gel matrix (12d).

95.. Method according to one of Claims 91 to 94, characterized in that a local temperature increase is set in a transverse region of the gel matrix corresponding to a starting region of the gel electrophoresis path.

96. The method according to any one of claims 91 to 95, characterized in that a laser arrangement (50, 52, 54, 56) is used to excite a plurality of fluorescent markers assigned to the molecules, the laser arrangement in the electrophoresis mode generating laser radiation which is at least one laser beam (60, 62, 64, 66), which runs essentially transversely to the direction of molecular propagation, cuts at least several of the gel electrophoresis tracks (12) in order to excite fluorescence markers of the molecules present in a detection area of the respective gel electrophoresis track, whereby A local temperature increase is set in a transverse region of the gel matrix that encloses the respective detection region.

97. The method according to any one of claims 90 to 96, that is applied to a device (10) according to one of claims 1 to 76 with a thermostatic plate (16b) through which a heat exchanger means can flow.

98. Method for the analysis of nucleic acids, characterized by the fact that products of nucleic acid sequencing reactions labeled with at least four different fluorescent markers are separated using a device (10) according to one of claims 1 to 76 and 83 to 89 according to their size and are analyzed independently of one another, if desired including the method according to one of claims 77 to 82 and / or the method according to one of claims 90 to 97.