WO2004046709A1

WO2004046709A1 - Method for analyzing molecules for molecule sequencing and spectrometer therefor

Info

Publication number: WO2004046709A1
Application number: PCT/DE2002/004275
Authority: WO
Inventors: Richard Fritz Sauter
Original assignee: Richard Fritz Sauter
Priority date: 2002-11-20
Filing date: 2002-11-20
Publication date: 2004-06-03
Also published as: AU2002363822A1; DE10297995D2

Abstract

The invention concerns a physico-chemical method for analyzing and sequencing molecules in a sample, said method using a spectrometer and a light beam of molecular thickness, called molecular spotlight for measuring transmission, diffusion and emission. The emission is stimulated so as to reduce the measurement time and to locate the emitting sequence position. This requires an intensive cooling device provided with several laser beams, fixed contacts for evacuating heat and fast flowing fluids, the quantum yield of the detection being enhanced by a special sensing and deflecting optical system. Maximum cooling of the amplifying optical system enhances the signal-to-noise ratio. The main fields of application of the invention are the key technology of molecular diagnosis, reading highly-compact stored data and polymeric analysis for exact determination tacticity and length of block copolymers and for measuring atomic-molecular progression lengths, even for macroscopic distances.

Description

description

Methods of analysis for molecules, for sequencing molecules and

Spectrometer therefor

Technical field

The present invention relates to a physico-chemical analysis method with associated spectrometer for molecules and sequence analysis of molecular samples.

State of the art

Nucleic acid sequencing was the central problem in genetic biochemistry. The previously established chemical-analytical processes process a maximum of around 20 bases per second and still require around six years for a human genome.

The DNA molecule of the longest human chromosome with about 3 * 10 base pairs is around 10 cm long. Other species sometimes have longer chromosomes. The diploid genome of a human body cell, as a single strand, is about 4 meters long and ^(' around 2 nanometers wide. The primary identity period, which, if at all, is only about 1.5% larger than that of the crystallographic c-axis of natural graphite with 0.3348 nm is 0.34 nm and the secondary is 3.4 nm. Every 0.34 nm is followed by a further position rotated by 36 °, here as one of usually four nucleotides, within the sequence of a sample The base of each nucleotide connected to it is the information-carrying part of a nucleic acid, and in contrast to the rest of the molecule, such bases can be determined optically as chromophores by spectral analysis.

The two base genera of puric acids can be removed from nucleic acids in a highly selective manner by means of an acid-catalytic polymer-analogous chemical reaction at p _H 3. This is also possible enzymatically, for example with the help of a nucleosidase. What remains is adenine- and guanine-free apuric acid. The sequence analysis of the primary and complementary strand, each with the remaining two base genera cytosine and thymine and uracil, complements Chargaff again for complete genetic information. The excitation of individual atomic elements with the tip of a scanning tunneling microscope to emit luminescence has already been achieved / EP 0 801 310 /. In this context, it is understood as a generic term for all phenomena of fluorescence and phosphorescence, since both can usually only be distinguished by the lifetime and the energy of their emission. Depending on the nature of the molecules, their temporal sequence is in the order of 10 ^" seconds for spontaneous fluorescence, typically in the range of seconds for phosphorescence

—14 phosphorescence, considerably reduced to 10 seconds / DE 101 23 443 /.

This is associated with a very high to maximum technically achievable space-time conversion of energy. Power lasers are therefore additionally cooled with water in order to achieve a thermal

-12

Avoid material decomposition. Pulsed operation is common. There are still no chemical reactions within 10 seconds, nor is there any energy transfer to neighboring positions by a molecular impact, in which the atomic nuclei are also noticeably moved. This is only partly due to the large volume expansion of the orbitals occupied by an excited electron as a standing wave of matter. According to the Bohr 'see postulates, calculated for the second shell of the hydrogen atom, this happens analogously at a speed in the order of magnitude of 1/300 of the vacuum light speed and takes around 10 ^" seconds, with a usual length extension of +100%

Biradical, one of which is particularly reactive. Furthermore, a change in the polarization between the excitation beam and the emission suggests that energy transfer has taken place. This means, above all at higher temperatures, that the glow mostly comes from a position other than the one just excited.

Overcoming disturbances of the measuring beam by the formation of streaks and gas bubbles has been state of the art for years. The Pentium 4 microprocessor is now operated at an operating frequency of 2.66 GHz, which is currently a monthly increase of +130 MHz.

"Light tweezers" and the tips of tunnel and force microscopes are suitable for numerous molecular micromanipulations of the sample. Documentation of the sequences, for example with the aid of electronic data processing, storage and output, is common. Presentation of the invention

The present invention solves the technical problem of specifying a method which allows an exact, reliable and economical sequence and molecular analysis to be carried out in a very short time. A key area of application is the key technology of genome analysis, for example for the purpose of molecular diagnostics. A second relates to the reading of miniaturized stored data or pits, for example in the field of extremely compact molecular storage density, and a third relates to the field of molecular trace analysis, for example for the detection of hidden explosives and intoxicants. Furthermore, this invention is useful in polymer analysis, for example for the exact determination of the tacticity of macromolecules and the block length in copolymers and also for high-precision, also macroscopic, length measurements with an atomic-molecular step size. The content of DE 101 23 443 is also part of this invention.

According to the invention, to solve the analytical problem, a thin light beam is generated in one of several ways, of which at least one dimension corresponds to the diameter of a molecule. That is the "molecular spotlight".

This can be a convergent beam with a small focal spot, a thin parallel beam and a preferably linearly polarized beam through a "picospalt". That is, a gap which is about 1 to 10 mm long and about as wide as the position distance of the sample. For nucleic acids it is 340 picometers. The electric field vector oscillates in particular in the longer direction of the "picospalt", so that no diffraction phenomena occur.

It can be manufactured e.g. by drilling and milling with an electron beam, if necessary, place several thin platelets with a gap on top of each other, as well as from plane-parallel separated and polluted corrosion-resistant metal pieces, which are provided with a molecularly thin spacer around the gap. The e.g. from graphene layers, mica platelets or metal sputtered on the edge.

The inventive method for sequence and molecular analysis is accordingly characterized by the features of independent claims 1), 7), 23), 24), 25), 27), 32) and 38). Advantageous refinements and developments are the subject of the claims which are directly or indirectly dependent thereon. The spectrometer according to the invention is given by claims 38) and 39). The inventive method for sequence analysis is characterized in that

A) the location and the size of the molecular position within the sequence of a sample are determined and recorded using a "molecular spotlight",

B) the individual sequentially consecutive positions within the sample, e.g. in the location determined in step A), are exposed to an electromagnetic excitation signal with the aid of a “molecular spotlight”, the one dimension corresponding to the position distance of the sample, in order to generate a response signal which is designed in particular as a characteristic light intensity such as transmission or luminescence,

C) the position from which the response signal originates is determined,

D) the respective response signal is detected, evaluated and stored, a detector device being used whose detection sensitivity is e.g. is in the range of a photon or electron,

E) the detection, the data processing and the advance to the next position are carried out within a relatively short time, in particular under one microsecond to under one nanosecond per position of the sample, and

F) steps B) to E) can be repeated at further positions within the sequence of the sample, preferably at their information-carrying parts.

A preferred embodiment of the method according to the invention is characterized in that the tips of tunnel or atomic microscopes are used to fix the sample.

According to a preferred embodiment, steps A) and B) can be carried out simultaneously, i.e. the measuring beam designed as a “molecular spotlight” serves on the one hand to determine the location and the size of a position and on the other hand it also causes characteristic absorption and Rayleigh scattering and the photoluminescence of the sample. In a further advantageous embodiment, the two steps A) and B) are temporal and carried out locally one after the other In an alternative embodiment, step B) can be carried out in a further pass.

When measuring the transmission, the spontaneous luminescence is preferably suppressed. This happens both with the stimulated emission and with a quencher, i.e. a substance that accelerates the quenching of the luminescence. Typical radical scavengers such as carbon tetrachloride, CCI ₄ , polyphenols such as hydroquinone and gases such as methane, CH ₄ are suitable for this. Electromagnetic radiation is suitable for excitation, i.e.: radio waves, microwaves, infrared (FR), visible radiation, ultraviolet (UV), X-rays and γ radiation, but also material radiation, such as electron beams, also in the form of alternating current and electrical fields. A system excitation and a system response in the area of optical radiation, that is from UV to FIR, are preferred.

The ionizing electromagnetic radiation types are applied so elastically, in particular by flat angles and radiation near the edge, that only an energy component required for characteristic transitions is transmitted to the sample, which is below the total energy 5 of the exciting photon.

In an advantageous embodiment, the optical excitation signal is designed so as to be characteristic of intensity, wavelength, spectral resolution, coherence length, polarization, phase and the duration of pulse and pause so that it is maximally absorbed by the part of the molecule to be excited. In particular, this takes place in a characteristic wavelength range and with a characteristic polarization, which is typical for the chromophore of the position of a sample just examined.

A preferred embodiment of the method according to the invention is characterized in that a stimulated emission is carried out in step B). This can also be done with an atomic molecular spatial resolution and in an internal environment, e.g. in the absence of oxygen,

5 happen and a transmission measurement can also be carried out. In addition, intensive cooling is carried out. This is done by flowing with a fluid, also supercritical, coolant, such as water, in particular at high speed. In addition, cooling can be achieved by contact with a cooling finger, which can also be designed as a wire. The material used for this is preferably characterized by a relatively high

0 Thermal conductivity and surface cleanliness. This is, for example, copper, silver and gold with an ion beam cleaned surface. Furthermore, cooling can also be carried out alone or in combination with a special laser beam with high spectral resolution. This is all the more effective if such radiation is carried out simultaneously from several different directions.

A scanning microscope can also be used for process steps A) or B). In an advantageous embodiment, the sample is water-free, undergoes rotation about its longitudinal axis, and deflection and collection optics increase the quantum yield of the detection. The working time for the system excitation, for the detection, the advance to the subsequent position and for the associated data processing preferably takes less than a nanosecond. With the known cryotechnology, a significant improvement in the signal-to-noise ratio in the amplifying electronics can also be achieved.

A particularly preferred embodiment, which is used in particular for the case of simultaneous excitation of a plurality of, in particular adjacent, positions, is distinguished in that the emission is evaluated via the appropriate use of difference spectra. This is also possible with the aid of "optical spreading". The position to be analyzed 5 is located directly in the focal point of a parabolic mirror. Adjacent sequences are outside of it. According to the rules of geometric beam optics, their luniinescence is not reflected parallel to the optical axis. A relative Long light path over several kilometers, for example with rolled-up fiber optic cables or plane-parallel special mirrors, enables spreading and thus also a lateral separation from the focal rays.

A further preferred embodiment uses several detector devices for the evaluation of the luminescence. This consists of the detector and upstream analysis units to refine the detection. The detector can be a photomultiplier, pulse height or multi-channel analyzer, a CCD camera, also pelet-cooled, or other fast and highly detection-sensitive photodetectors based on semiconductors, such as an avalanche

5 photodiode (APD) with a quantum efficiency of 50 to over 90%, also arranged as a diode row. In the infrared range, an MCT photodiode, camera and small sensor area are preferred for a faster signal measurement sequence.

In order to increase the accuracy or to achieve the highest possible lateral resolution, according to the invention an extra folying device can be used which acts on the excitation signals that are emitted. This consists of electric or magnetic field lenses as well as the "molecular spotlight" with an adjusted light intensity.

With emission in several directions, the space quantum yield is much better with the aid of a deflection and collection optics. This is, for example, a parabolic mirror, a part of it can also suffice, in the focal point of which the luminous position of the sample is preferably arranged. However, these 5 reflectors can also already contain the grating lines required for spectral decomposition, preferably of the echelet type, that is to say optimized in terms of efficiency, in particular on the focus of the wavelength of the radiation to be analyzed. If only the light intensity is measured, a sphere with a radius of 20 cm, for example, is sufficient, the inner surface of which is lined with relatively small detectors such as photodiodes.

In a particularly advantageous embodiment of the deflecting and collecting optics to improve the space quantum yield, a sphere with a radius of, for example, 5 cm is used with the light source in the center. In the spherical surface are exactly vertical, that is to say radial, the one ends of optical fibers which collimate the emission of the detector device from the other ends located at a common point

> feed. These optical fibers of different lengths are then preferably made thin and flexible, so their diameter is in the micrometer range. Their cross section is preferably planar and perpendicular to the length and is oriented towards full use of the spherical surface. That means, like a soccer ball, pentagons and hexagons alternate. Fewer optical fibers are required if the inner surface of the sphere that remains free is reflective

} is trained, e.g. as an aluminum mum ball. The radiation collected in this way is directed onto a detector device, in particular after a cross-sectional reduction with convergence, better with a pair of parabolic mirrors to maintain collimation.

Optical fibers can also serve as sample carriers, e.g. as a tube or hollow fiber filled with the sample, as a full fiber with the sample in a preferably central gap and with a sample fixed in casting resin 5.

Light sources are e.g. thermal, colorful and coherent emitters. One is required

9 14 20

Output from 10, 10 to over 10 photons per second, depending on which optical devices are irradiated with which loss up to the sample. Dye lasers are suitable for wide spectral ranges. Their spectral resolution can be improved) e.g. through tuning according to Fabry-Perot or Etalon as well as mode selection and mode coupling with electronic readjustment to a sharp spectral line. For pulsed excitation of the sample, e.g. electronic breakers, flash lamps and choppers are used. Reflectors can also be partially transparent, e.g. for preferably one-sided light passage, e.g. 1%.

5 A chopper separates temporally separate processes such as reflection and scattered light, stimulated emission, spontaneous fluorescence and phosphorescence, also to improve the signal / noise ratio. Instead of a chemical separation of the purine bases, to simplify the sequence analysis, the electron spin reversal stimulated by a characteristic microwave radiation is separated from their

) possibly also from the pyrimidine bases, excited states in relatively long-lived triplets. These then fail for around a second during regular absorption. The second electron, which is not excited, can also be stimulated with another characteristic microwave radiation to reverse the spin. If this happens, a sigulet state can no longer relax (so quickly) and separates from the regular one for so long

5 absorption also off. Characteristic J-R absorptions or Raman scatterings can also be measured, especially the stretching and bending vibrations of the base residues on the oxygen of the first carbon atom from the sugar.

The chemical improvement of the fluorescence intensity can also be achieved by photochemical derivatization with an organic reagent for the duration of the excited state. Hydroquinone, for example, is suitable for this.

The contact between the sample and the support can also be achieved using an adhesive, e.g. a household adhesive. It is also possible to extend the sample at one end. This happens with another polymer molecule that is chemically bound to it, for example via a condensation reaction.

5 Non-polar solvents such as ether or hexane, a glassy alcohol-ether matrix according to Spolsky at low temperature or high vacuum are also suitable as the chemical environment of the sample, with the advantage that the glass bulb of the detector tube, which may interfere with its reflection, can be omitted.

If the sample is excited with the tunnel current, it is advantageously to be measured water-free. 3 The strong electric fields of "naked" sodium ions then interfere, which must therefore be replaced by large-volume organic cations with a widely delocalized charge. For example, a quaternary organic ammonium salt is suitable for this.

Data storage is more compact if only one valid signal is saved at several measurements in the same position. Disturbing length fluctuations are achieved through a relatively high constancy of the working temperature through thermostatting, shading against external heat sources and use of materials with a particularly low expansion coefficient as well as a correspondingly low-vibration mounting of the spectrometer. This happens, for example, as with the tower microscope with a special suspension or how for a seismograph with a particularly heavy mass. "Low-leveP 'measuring station typical) precautions are the steel shielding to the outside, for example by the usual several cm thick lead walls, and within the spectrometer through the use of low-radioisotope materials.

The method for molecular analysis according to the invention is characterized in that

I) in principle the measuring beam of a conventional spectrometer for transmission and 5 fluorescence measurements with e.g. 2 mm diameter is replaced by the "molecular spotlight" as a molecularly thin measuring beam,

II) the detector of a conventional spectrometer is replaced by one with the highest possible detection sensitivity in the respective measuring range,

πi) the gain selectivity e.g. is operated in the lowest temperature range at temperatures around -196 ° C 3 to below 1 K (-272 ° C),

IV) the measuring beam is also matched to a maximum and selective absorption as possible according to the respective characteristic intensity, wavelength, spectral resolution, coherence length, polarization, phase and the duration of the pulse and pause, and

V) for gases and in solution, a square measuring beam is used.

5 A preferred embodiment of the method according to the invention is characterized in that a gas measuring cell, optionally with a heatable wall, is used in step I). In a particularly preferred variant, the light path in the gas measuring cell covers the entire Irmen cross-sectional area. The multiple total reflection of the measuring beam, even with several measuring beams, makes this possible, for example. The non-irradiated partial areas should not be significantly larger than one

0 measuring beam. Either its surface moves through the measuring volume or the gas flows through it. Instead of round e.g. also be square. This happens before the optical downsizing, if necessary after the measuring beam has been expanded, e.g. to 5 cm, with the help of an appropriately shaped panel. The measuring cell itself can also be square and reflective.

5 If the absorption by components of the carrier gas, usually air, becomes too large in a spectral measuring range required for characterization, this gas must be replaced by a gas, such as noble gas, for example argon, which has a weaker absorption therein. Interfering particles such as aerosols etc. are separated beforehand, eg electrostatically or by filtration.

As a solid, a molecule can also be examined optically in a capillary tube. Enrichment of the substance or group of substances sought is also advantageous. The characteristic strong absorption bands of organic nitro compounds in the IR spectrum at 1650, 1290 and 850 cm ^" and medium intensities are suitable for detecting explosives

760 and 700 cm. In contrast to sequence analysis, a significantly longer measuring time is available for determining a molecule. This can be less than a second, for example, but can also take up to an hour.

To create a parallel light beam with the usual 2 mm diameter with 200 p without disruptive diffraction as a "molecular spotlight", for example, a pair of parabolic mirrors with fl = 10 km and £ 2 = 1 mm, 3 identical pairs with fl = 1 m and £ 2 = 4.6 mm or 7 identical pairs with fl = 1 m and £ 2 = 10 cm are required .. If the light beam is reduced by more than 7 orders of magnitude, this can be done, preferably with the sample in a fixed position of the entire chromophore, only a single orbital can be spatially selectively excited.

In order to increase the spectral resolution, a copy of the sample, likewise a synthetic sample for molecular data storage, can be synthesized with more intense and faster-emitting, but preferably fluorescent, luminophores and then advantageously sequenced.

In order to direct every emitted photon onto the detector if possible, all components should be ideally transparent, absorbent and reflective, that is, each with a high degree of efficiency.

In addition to the “optical spreading”, a “picospalt” is also suitable for determining the position of the position under investigation within the sequence of a sample, which is also arranged in the beam path after the sample and is provided with an aperture to protect against radiation from adjacent positions. The transmission of the excitation beam also works in this sense. The same applies to the stimulated emission, both when stimulated with a “molecular spotlight” and in the resultant double emission. According to the invention, the photons specific for sequence positions are directed to a preferably pelet-cooled CCD camera after spectral decomposition in order to achieve the shortest possible measurement times, under one microsecond per sequence position. In the case of measuring times well below 0 nanoseconds, a fast electronic buffer with time-delayed data output, that is to say a transient memory, is used. The parallel operation of several microprocessors is also possible.

A preferred embodiment of the invention for sequence analysis has ten essential features:

5 1) An electromagnetic excitation of a sequence position with a lateral resolution in the order of magnitude from the distance between adjacent positions of the sample with the “molecular spotlight”,

2) stimulated emission at the same sequence position of the sample,

3) one, e.g. piezoelectronic, moving reflector device for directing the "molecular 0 spotlight" to the respective sequence position of the sample,

4) a similar reflector device for directing the emission onto the detector device,

5) highly effective cooling of the sample with multiple laser beams and heat dissipation contacts,

6) a relatively high detection sensitivity of the detector in the order of 5 photons,

7) a relatively short detection and data processing time under one nanosecond,

8) an interference-free power supply and shielded measurements e.g. in a Farady cage,

9) cooling of the amplification electronics e.g. in the lowest temperature range and

! 0 10) an image section the size of the longest sample.

Characteristic features of the invention in this method are that locally selective optical excitation of a sequence position of the sample using the “molecular Spotlights "takes place and for transmission and fluorescence measurements a detection of the resulting response signal is carried out in the range of highest possible detection sensitivity and shortest measuring time specific to the location and genus. The variant of the exciter spectroscopy technique can also be used for this.

The invention also relates to a spectrometer for performing the above-mentioned methods. There are devices or units which enable the method steps mentioned above to be carried out in a corresponding manner.

The principle of the invention is explained below with reference to the excitation of a sample to emit a luminescence.

Like any emission of electromagnetic radiation and of material radiation, luminescence can also be excited with the aid of both types of radiation. This is preferably done optically, but also with electrons. In principle, electric fields are also suitable for this alone, in particular in the form of alternating fields, and a sufficiently high temperature, which is, however, above the decomposition point of most organic substances.

Double-stranded molecules are preferred, in particular thermally, melted into single-stranded molecules and then individually examined. There is a possibility that the molecules to be sequenced e.g. be measured in a capillary with a light collecting effect.

The helical structure of the DNA macromolecules with m = 10 = primary / secondary identity period is also retained in the single-strand version. This means that the core bases follow each other rotated by 36 °. In order to measure them all from the same direction, a rotation around the longitudinal axis is required.

Refinement of the spectral decomposition with a monochromator device, e.g. With the help of a holographically generated grating with an optimized brightness due to the design, Fabry-Perot plates can be used to bring even higher spectral resolution.

According to the invention, the photons which are characteristic of a position of the sample and which, as is known, can originate from various excited species, are made as short as possible to achieve this

Measurement times directed to a common detector per position-specific genus. This can be a photomultiplier, as can a pellet-cooled CCD camera. To achieve a valid response signal, a position must be excited about two to one thousand times with the efficiency of this invention. Six detectors are sufficient for simple, quick and automatic determination of whether DNA or RNA is present, the proportion of rare special nucleotides and the exact degree of polymerization.

A preferred embodiment of the invention has five essential features:

1) a relatively high lateral resolution in the range of the minimum position distance of the sample, with nucleic acids = 0.34 nm,

2) a relatively high detection sensitivity of the detector in the order of magnitude of one photon,

3) a relatively high quantum yield from excitation to detection,

4) a relatively short detection time, e.g. below one microsecond to nanosecond,

5) an equally short time for a subsequent data processing step and

6) deep-freeze amplification electronics.

Description of the drawings

Figure 1 is a schematic representation of a spectrometer for absorption and fluorescence measurements for conducting a molecular and sequence analysis with a "molecular spotlight". It consists of: a light source (76), an attachment lens (96), e.g. a condenser, one

20

Control device for the optical intensity (81), for example from 0 to 10 photons per second, a monochromator device (78), a polarizer (80), a chopper (82), a parallel light beam with a diameter of approximately 2 mm, a first parabolic mirror (52 , 1) with focal length f 1 and focal point F 1, a second parabolic mirror (52.2), any number of further parabolic mirror pairs (52.3 + 52.4 etc.), a beam splitter (92) for branching off the reference beam (99) from Measuring beam (97), the sample (12) with the position just measured at the point “X” in a measuring cell or deflecting and collecting optics symbolized by the dashed circle, a chopper (82) for coupling in the vertical emission, a polarizer (80 ), a chopper (82) for coupling the reference beam, a monocliromator device (78), a polarizer (80), a control device for the optical intensity (81), a first detector (14 / Dl), a device for signal amplification (86) , one electrical line (32), a control, evaluation and storage device (18) and a data output unit (88). Between branching and coupling, the reference beam (99) is directed either to a third detector (D3) or to a deflection device (62). The same applies to the vertical emission to a second detector (D2) or to a deflection device (62).

FIG. 2 shows a freely movable arrangement of the sample carrier (22) consisting of two tunnel microscope tips (24 + 28) with the sample (12/40) hanging between them. The position just measured is then marked with "X". The excitation beam (97), the transmission (98) and the associated detel device (14) also follow. Furthermore, the radiation for the stimulated emission (34), the response from luminescence (30) also follows Rayleigh scattering and stimulated emission (36) and the associated detector device (14.) The x coordinate runs in the longitudinal direction of the sample, the y coordinate runs perpendicular to it and the z coordinate runs perpendicular to both.

Ways of Carrying Out the Invention

The following specific exemplary embodiment is described on the basis of the sequence analysis of DNA molecules. If they are in the form of a double helix, they are preferably melted using the thermal method. In each case one of the individual strands (40) of the sample (12) is fixed between two tips (24) and (28) of a tunnel microscope in a manner that is easy to handle and is thereby extended in the longitudinal direction (x) (see FIG. 2). Both tips are connected to each other via a bracket and can be moved freely in all directions as required.

With a "molecular spotlight" the sample is roughly examined for a first determination of the position (topography). The absorption, emission or scattering is measured. The light intensity lies below the saturation of the chromophore and the wavelength in the range from ER. To 230 nm. A noticeable absorption begins at λ = 310 nm. The coordinates (x, y and z) thus obtained are stored, advantageously setting y and z = 0 (upper edge of the sequence positions) and the first position starting at x = 0.

In the case of a further fine run, the position coordinates of the sample are determined and stored, or the excitation to emit a luminescence (30) takes place. Furthermore, both a separate second fine pass can be carried out and an excitation signal (20) can be emitted via a second “molecular spotlight” that follows immediately after it.

According to FIGS. 1 and 2, there is a spectrometer that generates a “molecular spotlight” and can direct it onto the fixed sample. It has a detector device (14), a deeply cooled electronic amplifier (86) and a control and evaluation unit. and storage means (18) followed by an output unit for the data.

The exact positioning of the sample in the spectrometer with regard to the location of its sequence position (X) is shown schematically in FIGS. 1 and 2 by the coordinate arrows x, y and z.

The position coordinate x (lassgsX Y (landscape) and z (portrait) of the respective sequence position (X) is first determined in a fixed manner. In the subsequent positions, the determination of the x coordinate is generally sufficient, since y and z usually remain constant at a value of 0. In a further run, the excitation signal (20) is then applied to the chromophore at this point (X) = n, whereupon the luminophore thus produced emits the luminescence (30), which is picked up by a detector device (14) and then is evaluated, stored and output by the data output (88) by the control, evaluation and storage device (18).

The diameter and thus the relatively high lateral resolution of the excitation beam (20/97) in the order of magnitude of typical positional distances within the sample is therefore 0.34 nm.

The following specific exemplary embodiment for sequence analysis is described using double-stranded DNA molecules. The sample is taken from a swab of the cheek mucosa from a person's oral cavity. This is followed by enzymatic digestion with lysis of the cell wall and nucleus, followed by isolation, separation and purification in one operation by accelerated gel permeation chromatography with a particularly short column, with negative pressure on the removal side and with centrifugal force. Duration about ¹ A to 1 hour, also automated. If the spectral purity of the sample is insufficient, this is followed by repeated cleaning if necessary, i.e. phenol extraction and precipitation from aqueous solution using isopropanol or ethanol. The double strands are then thermally separated into two complementary single strands by heating to over 65 to 90 ° C. in water. Possibly. add some glycerin or a mixture of sodium chloride and citrate.

PH 3 is adjusted at room temperature by adding phthalate-hydrochloric acid buffer. Possibly. ) reheat. Subject the mixture to gel permeation chromatography again or pour it into a dish, let it dry, rinse with pure water, let it dry again. Electrostatically, if necessary with biotin, fix the sample at two tips and place it in the holding device. Pull the tips apart without tearing the sample and place them in the measuring beam.

To check the position and size, the transmission, fluorescence and possibly the scattered light must be measured. To do this, start with one photon per second and then increase the intensity to saturation. With an emission stimulated with any lateral resolution, the saturation intensity is several orders of magnitude higher. The determinations become more precise when the photons that are superfluous for a measurement burden the detection. This can be achieved by the selective selection of a characteristic polarization; a correspondingly high spectral resolution of the excitation radiation is also advantageous. The phase also influences the success of the absorption of a light quantum. This can be moved cheaply by triggering the flash lamp or chopper. A matching extension of the light path is also beneficial. Heat dissipation contacts and the mostly multiple use of laser cooling dissipate disruptive heat.

ϊ Place position 1 of the sample in the measuring beam and measure the transmission at λ = 260 nm, likewise at λ = 230 nm. The measuring beam, which is hardly weakened in each case, indicates the absent bases adenosine (A) and guanosine (G). Thymine (T) shows the measuring beam which is hardly weakened at λ = 230 nm and significantly weakened at λ = 260 nm. If both times the measurement beam is attenuated approximately the same and clearly, cytosine (C) is present. The result is electronic

»Saved.

Piezoelectric, also precision mechanical or thermohydraulic, actuators move the sample holder relative to the spectrometer position by position to the last position after each successful measurement. After the limited maximum deflection of the actuator has been reached, the moving front part is fixed there, the rear starting part is set back to a minimum deflection, is then tracked, fixed and the fixing of the front part is released again. Alternatively, the actuators move a reflector that directs the measuring beam from the first to the last sequence position of the semicircular sample. When measuring the Transmission directs a second reflector, which is also moved, to a fixed detector. When measuring fluorescence, the sample is moved.

The complementary strand is sequenced in the same way and then the appropriate complementary direction and the complete sequence are determined with the aid of EDP. The result is then output as a complete sequence protocol or only in the form of special deviations as well as molecular genetic diagnosis and prognosis with therapy suggestions saved on a data carrier. This happens, for example, on a magnetic tape, a memory chip or a burned CD-ROM.

The following specific embodiment for molecular data storage is described with reference to DNA molecules synthesized according to the prior art. Letters and numbers are transferred according to the Morse code, dot and dash analogously, to the base sequence C and T of the nucleic acids as a molecular data store. These information units are read off as already described under sequence analysis, but without separating the purine bases.

Another example of reading stored data in conventional readers replaces the reading beam for the information units, also known as pits, with the "molecular spotlight", for example in the form of a thin convergent or parallel light beam. In this way, these pits can be made ever smaller.

The following specific embodiment for molecular analysis is described using Lysergic Acid Diethylamide. In conventional spectrometers for trace gas analysis, the too thick measuring beam is replaced by the "molecular spotlight" in the form of the thin, parallel and square light beam. The angle of incidence on the reflectors of the measuring cell with a volume of 1 mm ³ to 1 cm ³ is even flatter. Instead of a volume, only one surface is illuminated, even several times from all sides, which is either moved through the volume or through which the measuring gas flows. The characteristic and maximum IR absorption appears around 1620 cm ^" .

Characteristic features of the invention in this analysis method are that local excitation takes place in the atomic-molecular order of magnitude and at the same time a detection of the resulting specific response signal of the sequence position in the range of the highest possible detection sensitivity is carried out selectively from the location of the excitation. This enables simple, automatable, fast, inexpensive, direct and sequence-accurate analyzes of (macro) molecules and their parts as well as trace molecules. In particular, a human genome can be sequenced within a very short time, from less than a month to a quarter of an hour, at a cost that is compatible with health insurance.

Both the production of such spectrometers, also fully automatically, and their use in analytical services is possible, especially for gene, polymer and trace analysis.

Claims

claims

1) Analysis method for molecules and for sequencing molecular samples, characterized in that

+ A) the sample is excited with the aid of particle radiation to emit an analytical response signal, + B) the sample undergoes a rotation and

+ C) the sample is measured anhydrous.

2) Method according to claim 1), characterized in that a phase analysis of the particle stream is carried out as an analytical response signal.

3) Method according to claim 1), characterized in that the sodium ions of the sample are replaced by special cations.

4) Method according to claim 3), characterized in that the special cations are relatively large and have a widely delocalized charge.

5) Method according to claim 4), characterized in that the special cations are a quaternary organic ammonium compound.

6) Method according to claim 4), characterized in that the special cations are an organic crown ether compound.

7) Analysis method for molecules and for sequencing molecular samples, characterized in that

+ A) the sample is excited with the aid of electromagnetic radiation to emit an analytical response signal,

+ B) a "molecular spotlight" is used for this and

+ C) the sample is excited with a special characteristic quality.

8) Method according to claim 7), characterized in that the electromagnetic radiation is in the range of ultraviolet, visible and infrared light. 9) Method according to claim 7), characterized in that the electromagnetic radiation is in the range of characteristic wavelengths of the sample.

10) Method according to claim 9), characterized in that the characteristic wavelengths of a nucleic acid sample at 260 nm and 230 nm.

11) Method according to claim 7), characterized in that the special characteristic quality is so characteristic as intensity that it is maximally absorbed by the part of the molecule to be excited.

12) Method according to claim 7), characterized in that the special characteristic quality is so characteristic as a wavelength that it is maximally absorbed by the part of the molecule to be excited.

13) Method according to claim 7), characterized in that the special characteristic quality is so characteristically formed as a spectral resolution that it is maximally absorbed by the part of the molecule to be excited.

14) Method according to claim 7), characterized in that the special characteristic quality is formed as a characteristic coherence length so that it is maximally absorbed by the part of the molecule to be excited.

15) Method according to claim 7), characterized in that the special characteristic quality is so characteristic as polarization that it is maximally absorbed by the part of the molecule to be excited.

16) Method according to claim 7), characterized in that the special characteristic quality is so characterized as a phase that it is maximally absorbed by the part of the molecule to be excited.

17) Method according to claim 7), characterized in that the special characteristic quality as the duration of the pulse and pause is so characteristic that it is maximally absorbed by the part of the molecule to be excited.

18) Method according to claim 7), characterized in that the analytical response signal is a transmission. 19) Method according to claim 1) and 7), characterized in that the analytical response signal is a luminescence.

20) Method according to claim 19), characterized in that the luminescence is a fluorescence.

21) Method according to claim 18) to 20), characterized in that the analytical response signal is a stimulated emission.

22) Method according to claim 1) and 7), characterized in that the sequence emission position of the analytical response signal is determined.

23) Method for generating a convergent "molecular spotlight", characterized in that a parallel light beam and a non-aberrated parabolic mirror are used.

24) Method for producing a parallel “molecular spotlight”, characterized in that a parallel light beam and several non-aberrated parabolic mirrors are used.

25) Method for producing a polarized "molecular spotlight", characterized in that a polarized light beam and a "picospalt" are used.

26) Method according to claim 25), characterized in that linearly polarized light is used.

27) Analysis method for molecules and for sequencing molecular samples, characterized in that

+ A) the resulting response signal is electromagnetic radiation,

+ B) this emission is fed to the detection in a high quantum yield with an additional deflection and collection optics and

+ C) the sequence position from which the analytical response signal originates is determined.

28) Method according to claim 27), characterized in that the electromagnetic radiation is fed directly to a detector. 29) Method according to claim 27), characterized in that the electromagnetic radiation is fed to a detector after spectral filtering.

30) Method according to claim 27), characterized in that the electromagnetic radiation is supplied to a detector after spectral decomposition.

31) Method according to claim 27), characterized in that the deflecting and collecting optics is a paraboloid.

32) deflecting and collecting optics, characterized in that vertical optical fiber bundles are embedded in the surface of a ball.

33) Method according to claim 27), characterized in that the deflecting and collecting optics is a ball, the inner surface of which is lined with detector elements.

34) Method according to claim 27), characterized in that the determination of the sequence position from which the analytical response signal originates is carried out with the aid of the "optical spreading".

35) The method according to claim 34), characterized in that a relatively long compact light guide is used for "optical spreading".

36) Method according to claim 34), characterized in that plane-parallel special mirrors are used for the "optical spreading".

37) Method according to claim 27), characterized in that the determination of the sequence position from which the analytical response signal originates is carried out with the aid of the "picospalt" with an aperture which is arranged after the sample.

38) spectrometer for performing the method according to one or more of claims 1) to 37).

39) Spectrometer according to claim 38), characterized in that the detectors and microprocessors used are particularly fast at over 2.65 gigahertz.