DE102004037081A1 - Hybridization assay of nucleic acid, useful particularly for identifying nitrogen-fixing microorganisms, where the complex formed includes a lable in, or close to, the hybrid region - Google Patents

Abstract

Analyzing a sample by a ligand-binding process where target sequences (TS), present as part of taget molecules (I) form a duplex or complex (A) with capture sequences (CS) of probes (II) and/or where the resulting (A) are analyzed, is new. (A) contains, spatially close to and/or within TS, at least one detectable label or accumulation of labels. Independent claims are also included for: (1) preparing (I) that contain labels exclusively close to or within TS; (2) (I) for use in the new process where the (accumulation of) labels is close to and/or within, TS; (3) kit for preparing (I); (4) set of capture molecules for the new process; (5) detecting capture sequences; and (6) device for analyzing the nucleic acid from a nitrogen-fixing organism comprising a microscope slide on which capture molecules are immobilized, each having the same capture sequence as any of 189 sequences reproduced.

Description

The The present invention relates to methods for analyzing samples by ligand binding methods, target molecules used in such procedures and kits for providing such target molecules and / or execution the aforementioned method.

background the invention

Nucleic acids are usually in the form of double-stranded molecules which are also referred to as heteroduplexes and composed of two nucleic acid molecules are. If duplexes are subjected to a temperature increase, dissociate into two single-stranded ones Nucleic acid molecules. at lowering the temperature, these can become a heteroduplex reassozieren. The forward reaction to duplexes is called hybridization or Rehybridization. Duplexes are also called hybrids.

Nucleic acids are Polymers of the nucleotides adenine, cytosine, guanine, thymine, uracil, Inosine (a, c, g, t, u, i), artificial or modified nucleotides in the polymer such as beads a string of pearls are strung together. The sequence of nucleotides in A nucleic acid molecule is specific for the particular nucleic acid molecule. The formation of double strands over Hydrogen bonds, for example, between single nucleotides a and t, as well as c and g can arise, if enough on a single strand Nucleotides follow each other, which are based on a respective Gegennukleotid another single strand - and there in the appropriate order - meet. Come in nature nucleic acids in the form of such complementary single strands as Double strand before.

Frequently stands One faces the problem of having the presence of a particular nucleic acid in one Reaction mixture or a biological sample (hereinafter the term "reaction mixture" summarized) to be detected. This is usually done by the Use of catcher molecules, the single nucleic acids have, with a single-stranded or partially einsträngigien Nucleic acid molecule, below target molecule called hybridise to duplexes, and the detection of the duplex allows a statement about the presence of the target molecule. There on nucleic acids certain sequences can occur several times or occur several times, the catcher molecule is selected so that it preferably reacts with a target sequence containing a section represents the total sequence of the nucleic acid to be detected and for this preferably is specific. As a rule, one tries to achieve the highest possible stringency the catcher target sequence hybrid or duplexes and the greatest possible specificity or uniqueness the target sequence for the nucleic acid in question to ensure.

Detection of nucleic acids using capture-target sequence hybrids has been known for a long time. It has evolved from the so-called Southern blot over many variants to oligonucleotide chips or DNA microarrays, in which a few cm ^{2 of} thousands of oligonucleotide sequences are spatially separated as spots on a solid phase and act as catcher molecules. Many of these spots simultaneously allow for a qualitative and, to a limited extent, quantitative response to the presence or absence of particular sequences in a reaction mixture. There are also a variety of variations of the capture target sequence hybrid detection principle that are well known in the art.

Around a proof of a capture target sequence hybrid to enable you need one for this sufficient amount of detectable hybrids.

Frequently one confronts with small amounts of sample, which by means of a PCR reaction in several Cycles must be amplified. Every cycle of a PCR reaction is like copying, with the number of copies with each Step increases exponentially. Each step costs time and material and the further the copying progressed, the more erroneous can the copies fail.

ever fewer cycles are required to produce a detectable amount of sample to produce, the cheaper and more reliable Therefore, the hybridization experiment carried out with such a prepared sample becomes.

When quantifying hybrids, there are considerable uncertainties. The maximum number of hybrids in a probe spot or spot of a microarray may correspond at most to the number of capture molecules in the spot. This is generally known. On the other hand, how many of the catcher molecules of a spot form capture-target-sequence molecules in an experiment can hardly be reliably predicted. Several factors affecting hybridization efficiency are already known (Southern et al., 1999), such as the distance of the target molecule complementary sequence of the capture oligonucleotide from the glass surface. The steric effect was also described as a decrease in efficiency when the duplex leads to a long overhang of the target towards the glass surface (Peplies et al., 2003). The intensity of the detection signals, on the basis of which the Vorlie Although hybridization of hybrids appears to be approximately proportional to the number of hybrids present in a probe point, the proportionality factor does not appear to be identical from probe point to probe point if different hybrids are present at different probe points.

task The present invention is therefore a method for analyzing of samples by means of a hybridization, with that too very small amounts of sample can be detected more reliably and more clearly can than before, and in which the detected signals better in correlation can be set to each other as in known methods.

The Task is solved by a method of analyzing samples by a ligand binding method, in which binding of target sequences of target molecules with capture sequences generated by probes duplexes or complexes and / or in the thus generated Duplexes or complexes are analyzed using the duplexes or Complex in spatial Close to and / or within the target sequence a detectable label or a buildup of markings.

It has surprisingly demonstrated the use of capture sequences that are complementary to target sequences are the target molecules, the in spatial Close to it and / or within the target sequence a detectable label or a buildup have markings, lead in the inventive method to signal intensities, the across from Signal intensities, as in comparable methods using catcher sequences not selected according to the invention be achieved so that the mark is not in spatial Close to Target sequence is significantly higher.

Preferably is the detectable label or the accumulation of labels exclusively in spatial Close to and / or within the target sequence.

Target molecules can with be marked with a single mark. In principle, however, it is also common to target molecules with several To provide markings. Here is the accumulation of marks in spatial Close to and / or within the target sequence.

When accumulation of markings in the context of the present invention is preferably the largest accumulation of Markings on the target molecule understood, which is located within an area that is not longer as the target sequence, allowing for specific duplex formation. in principle Is it possible, that at the target molecule further markings or accumulations markings are provided, but they are not always located in areas the for the target molecule are specific and therefore are not suitable as a target sequence.

Thereby, that the target sequence on for the target molecule limited to specific areas is often not freely variable, which is why according to the invention both at least one marker close to or within one for the Target molecule specific Provision is to be made and the capture sequence is complementary to this Range, which then represents the target sequence.

at a method according to the invention several samples simultaneously analyzed by ligand binding, where all target molecules at least one detectable marker in spatial proximity to or in the respective one Have target sequence.

To a further inventive method assign all target molecules the same number of marks on.

In inventive method be at least ten samples, preferably at least a hundred samples or at least a thousand samples analyzed simultaneously.

In inventive method For example, the label may be a fluorescent label.

In inventive method the fluorescent label is determined by one or more of the following Marking Agent: Cy3, Cy5, Fluorescein, Texas Red, Alexa Fluorine Dyes and / or other fluorescent dyes.

The Task is further solved by a method of producing target molecules which in spatial Close to respective target sequence or within the respective target sequence have at least one mark.

at The invention becomes target molecules uses which target sequences and a marker in spatial Close to or within the respective target sequence.

These target molecules can be used in the previously described inventive methods and lead to capture-target sequence hybrids which have a higher signal intensity than just those hybrids which are generated with the aid of target molecules which have target sequences and a label for the respective target sequence sen, which is not in close proximity to this.

The Labeling of the target molecules can be detected by enzymatic end-labeling, reverse transcription, indirect labeling and / or "sandwich" methods.

The Task is further solved by a method for generating target molecules by means of a PCR method, in which at least one primer provided with a marking agent is used.

In the method according to the invention can be at least another with an identical or another Labeling primed primer can be used. To this In this way, different types of signals can be provided in order to obtain results in inventive method to verify, but also to the intensity of different spots, in which have different numbers of hybrids, to each other same, so the intensities in the same order of magnitude lie. The spot in which more hybrids can be present, for example have the marking agent with the lower signal intensity. Because the signals are due to different labeling means differentiate from each other and the factor around which theirs signal intensities differ from each other, is known, allows such a method a quantification and a better comparison of the intensities in both Spots. This is particularly advantageous if the use one higher signal intensity having marking agent to a saturation value in the detection device, about a photomultiplier, would lead. Then would be the same one Quantification due to technical conditions not possible or at least more difficult.

Puts in a PCR method, primers that are labeled and dNTPs, the above have no mark, so the PCR product is at the end formed by the primer is marked, and on the others Do not mark marks. Chooses the primers so that the target sequence of the PCR product is in spatial Proximity to Primer, the PCR product is a target molecule that is exclusively available in spatial Close to Target sequence a marker according to the invention having. This is a very simple and uncomplicated process, to arrive at target molecules of the invention. By using defined primers with a certain length, let yourself a more precise statement about it make how much labeling agent incorporated into each one target molecule was as if a mix of marked and unmarked dNTPs is used to generate a labeled PCR product; because if individual dNTPs are marked, that is not always the case clearly how many labeled dNTPs are incorporated into a PCR product, and how many unmarked ones are incorporated therein, or the PCR products differ with regard to the number of labeled markers incorporated therein Nucleotides. It is also from depending on the amplified sequence. Usually one of the four Marked bases marked. Dip these in an amplification product less often on, so contains the amplification product will contain less labeling agent than other amplification products. This circumstance makes the meeting much more difficult Statements based on a microarray experiment. By an appropriate Primer selection leaves make sure that the primers used in an experiment each have approximately the same amount of marking agent. These Quantities no longer vary depending on a target sequence to be amplified, but hang solely by the structure and the design of the primer used in each case from. Apart from the raised Signal intensities, which significantly improve the sensitivity of microarray experiments, represent inventive method for the reasons mentioned also much more accurate than in the prior art known method.

at a method according to the invention for the production of target molecules according to the invention are targets generated by a PCR method, and it is doing at least used a kind of dNTPs that provided with a marker are, with the labeled dNTPs in close proximity to the target sequence and / or incorporated into the target sequence itself.

in the A difference from the prior art is in such a method according to the invention ensure that labeling agent is in close proximity to the target sequence indeed occurs. Close to the target sequence in this context means that labeling agent to nucleotides incorporated into the target sequence or to directly adjacent the target sequence incorporated nucleotides is bound. In immediate Close to Target sequence in the sense of the invention means that the marking or that the labeling agent is not more than 100 or 60, preferably not more than 0 - 20 Bases away from the target sequence. Significant positive effects (Factors 2-146) still be detected at a distance of 100 bases. Preferably the tag is exclusively in close proximity to the target sequence arranged.

Further methods according to the invention include the fluorescence labeling of nucleic acids (DNA or RNA) by means of other methods, for example by enzymatic end-labeling (such as, for example, by terminal transferase, polynucleotide kinase, Po ly (a) polymerase or other enzymes) or by reverse transcription with fluorescently labeled primers, or by indirect labeling methods.

One Another inventive method for Preparation of target molecules according to the invention is a Method in which target molecules be generated by a PCR method in which at least one Type dNTPs are used, which are provided with a marker are in which the labeled dNTPs or an accumulation thereof in the immediate Close to Target sequence and / or incorporated into the target sequence itself, and in which one or more primers is used or used be the one or the other with the same or further markers is or are provided.

Further inventive method for the preparation of target molecules according to the invention are methods, in which labeling agents in close proximity to the target sequence and / or be incorporated into the target sequence itself.

Here many processes are possible e.g. Incorporation of labeled dNTPs into cDNA by reverse transcription, or post-labeling protocols that utilize the incorporation of aminoallyl-nNTP into cDNA and then a chemical coupling with fluorescent dyes, or direct non-enzymatic labeling of the RNA with fluorescent dyes.

One such procedure leads to target molecules of the invention, the even at very low concentrations of formed hybrids still good are detectable, since by the incorporation of marker in the Target sequence comparatively much labeling agents are incorporated can. At least for target molecules present in normal concentrations on the basis of the further marking agent in the primer used the Result in a spot as well be verified.

The Task is further solved through the target molecules for use in methods according to the invention, the one mark or an accumulation of markers in spatial Close to or within the respective target sequence.

Prefers are by the method according to the invention provided target molecules according to the invention.

The Task finally becomes by a method according to the invention solved, used in the target molecules according to the invention which also come after a process according to the invention for the preparation such molecules may have been produced.

• – eine vorbestimmte Anzahl unterschiedlicher Fängermoleküle umfassend jeweils unterschiedliche Fängersequenzen, die jeweils zum Ausbilden von Ligandenbindungen mit Zielsequenzen von erfindungsgemäßen Zielmolekülen derart ausgebildet sind, dass sie komplementär zu den jeweiligen Abschnitten der Zielsequenzen sind, die sich in räumlicher Nähe zu einer Markierung bzw. einer Anhäufung von Markierungen befinden.

A set of catcher molecules according to the invention for carrying out processes according to the invention comprises:

• A predetermined number of different capture molecules each comprising different capture sequences, each designed to form ligand bonds with target sequences of target molecules of the invention such that they are complementary to the respective segments of the target sequences that are in spatial proximity to a label or an aggregate of Markings are located.

One inventive sentence Catcher molecules points at least 10, 30, 50, 100, 1,000 or 5,000 different capture sequences on. Preferably, all catcher molecules of the set are Catcher molecules like this trained to be complementary to the respective sections of the target sequences, which are in spatial Close to one Marking or accumulation of markings.

at a sentence according to the invention are the catcher sequences Components of the probes of a DNA array.

To a method according to the invention the catcher molecules in such a way determines or calculates that they are complementary to the respective sections the target sequence, which are in proximity to a mark or to an accumulation of markings. The target sequence here is an area of the target molecule definitely, for the target molecule is specific.

Precise description the invention

The The invention will be described below with reference to the figures and some embodiments explained in more detail. The Figures show in:

1a schematically a 362 bp target sequence according to one of the embodiments,

1b a table in which oligonucleotides are listed, as used in the embodiments,

1c a table in which oligonucleotides are listed, as they are used as primers for PCR amplification in the embodiments,

1d schematically the model system,

2a Fluorescence signals of hybridization of a Cy5-labeled antisense strand with sense oligonucleotides,

2 B a schematic representation of the hybrid molecules,

2c a graphical representation of detected signal specificities,

3a Fluorescence signals of hybridization of a Cy5-labeled sense strand with antisense oligonucleotides,

3b a schematic representation of the 3a belonging to hybrid molecules,

3c a graphic representation of the signal intensities of the fluorescence signals 3a .

4a Fluorescence signals of a hybridization of a Cy3-labeled sense strand with antisense nucleotides,

4b a schematic representation of the hybrid molecules 4a .

4c a graphic representation of the signal intensities of the fluorescence signals 4a .

5a Fluorescence signals of a hybridization of a Cy3-labeled antisense strand with sense oligonucleotides,

5b a schematic representation of the hybrid molecules to the fluorescence signals in 5a to lead,

5c a graphic representation of the signal intensities of the fluorescence signals 5a .

6a Fluorescence signals of the hybridization of a Cy3-labeled sense strand with antisense oligonucleotides,

6b a schematic representation of the hybrid molecules, the fluorescence signals according to 6a to lead,

6c a graphic representation of the signal intensities of the fluorescence signals 6a .

7a a schematic representation of 50mer oligonucleotides according to the embodiments,

7b a table of the 50mer oligonucleotides 7a .

8a Fluorescence signals of the hybridization of a Cy3-labeled sense strand with 50mer antisense oligonucleotides,

8b a graphic representation of the signal intensities of the fluorescence signals 8a .

9a Fluorescence signals of the hybridization of a Cy3-labeled antisense strand with 50mer sense oligonucleotides,

9b a graphic representation of the signal intensities of the fluorescence signals 9a .

10a Fluorescence signals of hybridization of fluorescein-12-dUTP labeled sense strand with short antisense oligonucleotides, which strands are not end-labeled but are internally uniformly labeled by incorporation of fluorescein-12-dUTP,

10b graphical representation of the signal intensities of the fluorescence signals 10a .

11a Fluorescence signals of hybridization of a Cy5 end-labeled sense strand with short antisense oligonucleotides as in 3 .

11b a graphic representation of the signal intensities of the fluorescence signals 11a .

12a Fluorescence signals of hybridization of a Cy5-end-labeled, 5 'end truncated sense strand with short antisense oligonucleotides as in 11 , and

12b a graphic representation of the signal intensities of the fluorescence signals 12a ,

Of the Invention is the surprising Findings based on the signal yield in fluorescently labeled Target molecules, the with catcher sequences Form hybrids or duplexes, higher are when the fluorescent label is near the hybrid formed lies. Unexpectedly, this effect is extraneous and thus sequence independent.

Surprisingly the observed effect is also independent of the chemical nature of the Fluorescent label. The effect occurs both when using long (eg 50mers), as well as short capture oligonucleotides (e.g. 16-17 mere). Surprisingly this effect is as well independently from the glass microarray surfaces or coating used Immobilization of capture oligonucleotides.

Based the following embodiments let yourself better explain the invention. The embodiments make it clear that the invention is a dramatic improvement the signal yield in and the significance of microarray hybridization experiments leads.

1D schematically shows the structure of a A duplex or hybrid complex A produced by means of a method according to the invention is bound to the surface B of a DNA array, for example a glass surface, via a spacer C, for example a poly adenine section of an oligonucleotide, a capture sequence or a capture oligonucleotide D. Complexed by a target sequence E is a target molecule F with a position 1, in 1D denoted by G. This is the first base of the hybrid complex A from catcher sequence D and target sequence E. A fluorescent label H is located thereon. The hybrid complex shown schematically represents an embodiment of the invention in which the label H is incorporated within the target sequence F of the target molecule E. ,

The Invention leaves on the basis of the following embodiments better explain.

The now following embodiments show that the invention the signal intensity in microarray experiments significantly improved. This is shown by means of certain fluorescent labels. The embodiments demonstrate that it is essentially the position of the tagging agent arrives with respect to the hybrid to be detected. It is perfect here no matter what a marking agent in detail. It only comes and only on the position of the tagging agent in relation to the to be demonstrated hybrid or duplex, of course, mixed effects with others Factors that may affect hybridization efficiency (e.g. Length of the Oligonucleotide spacers, steric effects, secondary structure effects), occur. The following embodiments are therefore only illustrative examples to understand. In particular, restrict the following embodiments and that just explained Do not experiment with the teachings of the invention with a view to be proved Sequences, labels to use or the like.

EXAMPLES

The embodiments are components of an example experiment, which was designed as follows:
The target molecule in the example experiment is a 5'-end-labeled or by random labeling with fluorescein-12-dUTP labeled single-stranded DNA, in the fragment of the nitrogenase gene nifH (Hurek et al., 1995) from the bacterium Azoarcus sp. Strain BH72. The fragment was amplified by PCR with the primers Zehr-nifH from chromosomal DNA of strain BH72 (Hurek et al., 2002), one of the primers being labeled with Cy3 / Cy5, the other with biotin. Fluorescence-labeled single-stranded DNA could thus be isolated from the PCR product. Using fluorescein-12-dUTP for random labeling, only biotinylated primer was used. The primers had the in 1C listed sequences, primers Z-nifH-f and Z-nifH-r (Zehr and McReynolds, 1989), except for experiments related to 11 (Primer Z-nifH-f-BN72-Cy5 and Z307-nifH-r-BH72-biotin) and 12 (Primer Z114-nifH-f-BH72-Cy5 and Z307-nifH-r-BH72-biotin) have the following sequences (Zehr and McReynolds, 1989):
Biotin-labeled strands were separated by streptavidin-coated paramagnetic beads (Roche) (Niemeyer et al, 1999). The concentration of the remaining single-stranded DNA was determined spectrophotometrically. Prior to each hybridization, the single-stranded DNA was denatured at 95 ° C for 10 minutes and then incubated on ice for at least three minutes.

The oligonucleotides used in this experiment all bind to the above mentioned nifH gene fragment of strain BH72. The corresponding sequences and their characteristics are shown in Tables 1 in 1b and 2 in 7b , A schematic representation of a possible duplex after hybridization gives 1D , Oligonucleotides bearing either 5 'amino modifications (amino link c6) or 3' modifications, some carrying poly-A spacers of 6-12 nucleotides, were synthesized by Thermoelectron, (Ulm, Germany).

to execution The hybridization experiments were performed on standard micrographs using DNA microarrays Glass slides made by Menzel, Braunschweig, Germany. chemicals and solvents came from the company Fluka (Neu-Ulm, Germany). For the production In the microarrays, the glass substrates were cleaned, silylated, and activated as described by Bentas et al (2002). The activated surfaces were directly immobilized to either 5'- or 3'-amino-modified Capture oligonucleotides used by covalent bonding.

The Application of the probes on the so activated slide surfaces was carried out using a piezo-driven spotter Robodrop (BIAS, Bremen, Germany) or by means of a MicroGrid II Compact 400 from BioRobotics, United Kingdom. The concentration of oligonucleotides was about 10 μM per ml Water. The water used contained 1% glycerin. In every spot of the microarray, about 250 pl were applied, which is a spot diameter of about 200 μm equivalent.

The slides were incubated overnight at room temperature in saturated aqueous humor to effect covalent attachment. The microarrays are blocked by means of 6-amino-1-hexanol (50 mM) and diisopropylethylamine (150 mM) in dimethylformite according to Beier et al (1999). Subsequently, the slides were washed with deionized, particle-free water, air-dried and stored at 4 ° C. under N ₂ .

The Hybridize the target molecules to the probes of the microarray and washing took place in a staff Hyb kiln of the company Stratagene, United States of America. The Duration of hybridization was from 1 to 16 hours. If not stated otherwise, the hybridization was carried out at room temperature with 50% formamide, at 46 ° C with 50% formamide, resulting in 10 nM single-stranded DNA used. The hybridization buffer used contained 4x SET, 10x Denhardt's. During hybridization became the slide covered with a coverslip. Following hybridization occurred a wash with 2 × SET (0.1% SDS) for 5 min and 1 x SET (0.1% SDS) for For 10 min at room temperature, or with 1 x (0.1% SDS) for 5 min and 0.1 × SET (0.1% SDS) for 10 min at 46 ° C. The dried microarrays were at a resolution of 10 μm with a GenePix 4000 microarray scanner from Axon, Union City, California with consistent power of the laser and at the same time Sensitivity of the photomultiplier evaluated. For this reason let the signal intensities determined in the respective exemplary embodiments be compared.

Embodiment 1

The reverse complementary strand or antisense strand of the nifH gene fragment of strain BH72 already mentioned above was hybridized with the sense oligonucleotides S307 (6A), S114 (6A) and S20 (6A). The antisense strand is in 1A shown schematically. The Cy5 label was introduced into the strand using a Cy5-labeled primer to generate the strand. In 1A schematically show the binding sites of the sense oligonucleotides on the antisense strand, the respective distance of these binding sites from the 3 'or 5' end of the antisense strand are shown. The antisense strand represents the target molecule, and the sense oligonucleotides represent the capture oligonucleotides or capture sequences, respectively, which have been applied in the form of probes on a microarray. The hybridization of these probes with the target molecule leads to different signal intensities in the respective spots on the microarray, as in 2A are shown. From left to right, these spots contain in pairs catcher oligonucleotides, respectively, having the sequence S307 (6A), S114 (6A), and S20 (6A). The antisense target strand is only labeled at the 5 'end. Spatially closest to the 5 'end is the binding site or target sequence S307 (FIG. 6A). Hybrids formed at this point are in the first two in 2A demonstrated spots. The signal intensity is highest there. Farther from the mark is the sequence section S114 (FIG. 6A), which in the next two spots in FIG 2A is detected. The signal is much weaker there. Slightly stronger, but still weak, is the signal that hybrids emit, involving the binding site S20 (6A).

In 2 B are schematically shown the capture oligonucleotides 1 . 2 and 3 as well as the target molecule 4 in the form of hybrid molecules consisting of 1 and 4 . 2 and 4 , and 3 and 4 and the respective resulting position of the marker 5 at the target molecule 4 in each hybrid. The capture oligonucleotide 1 corresponds to S307 (6A), the catcher molecule 2 S114 (6A), and the capture molecule 3 S20 (6A).

In 2C the corresponding signal intensities are shown graphically. It can be clearly seen that the use of the capture oligonucleotide S307 (FIG. 6A) or 1 leads to hybrids in which the label is in the immediate vicinity of the hybrid, and thus has a signal intensity that is 3 to 7 times higher than the other hybrids shown is achieved. It can also be seen that a particularly great reduction in the signal yields is present when the fragment to be hybridized is bound to the capture nucleic acid far from the labeled primer (S114 (FIG. 6A)).

Embodiment 2:

This embodiment shows that the effect described in Embodiment 1 is stranded and thus sequence independent. Cy5-labeled counterstrands (sense strand) were hybridized with the corresponding antisense oligonucleotides. The same effect was observed as in Example 1 (cf. 3A - 3C ). In 3A are each from left to right four spots with identical capture oligonucleotides, with left to right spots with the capture oligonucleotides A20 (12A), A20 (6A), A20 (6A) 3 ', A307, A307 (12A), A307 (6A), A307 (6A) 3 ', and A114 (6A). In 3B is shown schematically in the same order as the target molecule 7 that a mark 8th at its one end, binds to the respective oligonucleotide, and the resulting arrangement of the label with respect to the hybrid formed 9 is in 3B recognizable.

As shown by the graphical representation of the signal intensities in 3C , but also based on the brightness of the spots in 3A can be seen, the signal intensities are always highest when the marker is in spatial proximity to each formed hybrid, in comparison to the other hybrids by twice to a bit 4½ times, in extreme cases, even far above hi (see the values for A307 (6A) 3 'in 3C ). The designation 6A or 12A (the capture oligonucleotides) denotes the length of the respective spacer. This embodiment shows that the difference in the length of the spacer, ie the spacer between the glass surface of a microarray and the binding region of the capture oligonucleotide, has a comparatively small effect on the signal intensity. Thus, the difference between A20 (6A) and A20 (12A) is not particularly pronounced, and there is almost no difference between A307 (12A) and A307 (6A). A slight lowering of the signal at A20 (6A) 3 'compared to A20 (6A) could be caused by quenching effects due to the large spatial proximity of the fluorescent dye to the glass surface.

It It is noted in this context that in microarray hybridization experiments false negative results can be caused by a marking yourself in an unfavorable Position that leads to a low signal intensity, such as for example, in the case of the capture oligonucleotide A307 (6A) 3 '; similar steric effects of hybridization were reported by Peplies et al. (2003) described. This oligonucleotide provides a 48-fold lower signal intensity as A20 (6A). The worst Position is far from the target sequence.

Embodiment 3

This embodiment shows that the effect observed in the preceding embodiments can be further enhanced by closer proximity of the tag to the target sequence. An antisense scavenger oligonucleotide was used for this, A1 (6A) in 11 , which is complementary to the primer oligonucleotide, was generated by PCR, the end-labeled probe. As a result, the fluorescent label is already positioned at the first nucleotide of the heteroduplex that results from hybridization. In this embodiment, the fluorescence signal is increased by a factor of 2.3 compared to a 20 nucleotide-shifted capture oligonucleotide, A20 (FIG. 6A). How out 11A and 11B As can be seen, for intermediate positions (A4 (Fig. 6A) - A14 (Fig. 6A)), the signal strengths are descendingly weaker than for A1 (Fig. 6A). Compared to the worst case position of Embodiment 2, A114 (FIG. 6A), even a 146 times stronger signal is now observed at the most favorable position A1 (FIG. 6A). An even more distant but still relatively central position A188 (FIG. 6A) leads to a still low signal intensity (factor 357). The embodiment proves that relatively high signal yields can be obtained if the label is located less than 64 nucleotides from the target sequence forming the heteroduplex, in this embodiment there is already a 15-fold weaker signal.

The positive position effect is also in 12 clarified. If a truncated labeled probe is used (here by 113 nucleotides, so that the primer used for the PCR is complementary to the capture nucleotide A114), a similar positional effect for other capture oligonucleotides is shown. Oligonucleotide A114 (6A), which is described in U.S. Pat 11 in the middle position only low signal strength (130 rel intensity), shows in 12 as a terminal capture oligonucleotide high signal strength (11,800 rel intensity). Again, in this example, a strong decrease in signal intensity (factor 5.2) is observed when the label is at a greater distance (eg 74 nucleotides for A188 (A6), 12A , B) to the formed hybrid.

Embodiment 4

This embodiment shows that the effect observed in the previous embodiments is independent of the chemical nature of the label. The same experiments as in Embodiment 2 were carried out with a Cy3-labeled sense strand instead of a Cy5-labeled sense strand, and gave substantially the same results as described in Example 2. These results are in the 4A - 4C shown, with the marks in 4B with the reference number 10 , and the target molecule thus labeled by the reference numeral 11 is provided. In 4A each four spots are shown from left to right, in which catcher oligonucleotides are as described in Example 2. In 4B is shown schematically from left to right, as the respective capture oligonucleotide hybridizes to the target molecule, and in which position the marker is in relation to the hybrid formed in each case. 4C shows the signal intensities of in 4A shown spots in the same order.

As already indicated, the results essentially correspond to the in the embodiment 2 discussed results. That another mark too substantially results in the same results, proves that it does not matter what kind of fluorescence label chosen is to carry out the invention.

Embodiment 5:

The experiment described in Example 1 was repeated with Cy3-labeled sense strand and Cy3-labeled antisense strand. The results in the case of the Cy3-labeled sense strand are in the 5A - 5C which are the results of the Cy3-labeled antisense strand experiment in the 6A - 6C displayed. The signal intensity is highest where the marker is 8th in close proximity to each formed hybrid 9 located. When using the Cy3-labeled sense strand as the target molecule, this is the case in the spot with the capture sequence for S307 (6A), and in the Cy3-labeled antisense strand in the spot with the capture sequence for A20 (6A) (cf. 5A - 5C . 6A - 6C ).

These with the embodiment 1 comparable experiments show that both Cy3-labeled Sense strand as well as Cy3-labeled antisense strand then with high Signal yields are detectable when the hybrids formed with capture sequences have the mark in the immediate vicinity. For the A20 probe (6A) or S307 (FIG. 6A) is the signal intensity over other hybrids up to increased by a factor of 22.

Embodiment 6:

This embodiment demonstrates that the invention also works when using longer oligonucleotides. In this case, 50mer oligonucleotides were used, each at the outer ends of the target molecule tie. Cy3-labeled sense strand shows the stronger signal when the marker near the duplex (A19-68). The signal intensity was in in this case the rest Signals increased by a factor of 2.

Designation and sequences of the 50mer oligonucleotide scavengers or target sequences used can be found in Table 2 in 7B The position of the corresponding binding sites on the sense or antisense strand of the 362 bp nifH gene fragment from strain BH72 are shown schematically in FIG 7A shown.

In 8A the spots of a corresponding microarray are shown, with six identical spots in each row. The detectable in the respective spots target sequences or catcher sequences are in the presentation of the 8A on the right side of the illustrated microarray surface. In 8B The correspondingly determined signal intensities can be found in the form of a graphical representation.

The same experiment was performed with Cy3-labeled antisense strand. Also in this case, the strongest signal was when the label was near the duplex (S289-338), cf. the 9A . 9B , Again, the signal intensity was increased when performing a method according to the invention over the other signal intensities by about a factor of 2, in any case significantly improved, as from 9B for sequence S289-338.

Embodiment 7:

This embodiment demonstrates that other labeling strategies can be used to generate target molecules. Using unlabeled PCR primers and the random incorporation of fluorescein-12-dUTP (random labeling), a suitably labeled sense strand was generated that was hybridized with short antisense oligonucleotides 10A four grid points or spots are shown on the left in a raster consisting of rows and columns. The rows are numbered from I to IV and the columns labeled a - e. To the right is a schematic representation of the same grid, wherein the designation of the catcher oligonucleotides located in the respective spots is entered in the corresponding grid areas. For example, in panel IIa, spots with catcher oligonucleotides A307 (Fig. 6A), etc. are located 10E the signal intensities determined for the respective spots are shown graphically. The signal intensities are highest in the spot A20 (Fig. 6A) 3 'followed by the spot A20 (Fig. 6A) and A20 (Fig. 12A).

From Table I in 1B It can be seen that the capture oligonucleotide A20 (Fig. 6A) binds 3 'at four positions to T and U, respectively, while the capture oligonucleotide 307 binds to 0 positions at T and U, respectively, so that a higher incorporation rate of fluorescently labeled nucleotides in the A20 target sequence. As a result of fluorescence labeling on these bases, a higher fluorescence intensity is present in duplex at A20 than at A307, which results in a noticeably higher signal intensity (cf. 10B ).

In addition to the above-described glass slides commercial carrier for Micro-arrays were used, such as aldehydes and amine Slides slides, aldehyde plus slides the company Genetics, QMT ^® slides from aldehydes Peqlab, Pan amines ^® slides from MWG-Biotech. In these microarrays, the same results were found as just explained with reference to the embodiments 1-7.

This proves that the present invention in connection with any Microarray is usable.

above was labeled according to the invention Target sequences in solution applied to DNA arrays, demonstrated that the invention strand and thus sequence independent is.

In the context of the invention, this procedure is readily reversible, ie it can be Provide target molecules to be examined on an array, which are added according to the invention labeled, in solution catcher molecules, so that duplexes or complexes with high signal intensities arise. In other words, the term "target molecule" should be read as being interchangeable with the term "capture molecule" and vice versa. At the same time, the term "target sequence" is read in such a way that it has to be exchanged for the term "capture sequence" and vice versa.

Also let yourself in the context of the invention, the entire ligand binding reaction is basically accomplished in solution.

Bibliography

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Claims (26)

Method for analyzing samples by means of a ligand binding method in which, by binding of target sequences of target molecules with capture sequences of probes, duplexes or complexes are generated and / or duplexes or complexes are analyzed, characterized in that the duplexes or complexes are in close proximity to and / or within the target sequence have at least one detectable label or an accumulation of labels. Method according to claim 1, characterized in that that the duplexes or complexes exclusively in close proximity to and / or within the Target sequence at least one detectable label or an accumulation of Have markings. Method according to claim 1 or 2, characterized that a catcher sequence which is complementary to a target sequence in which spatial Proximity and / or within this target sequence at least one detectable label or a buildup located from markings. Method according to one of claims 1 to 3, characterized that multiple samples are simultaneously analyzed by ligand binding be, with all target molecules either at least one detectable label or an accumulation of Markings in spatial Close to have the respective target sequence. Method according to one of claims 1 to 4, characterized that all target molecules have the same number of marks. Method according to claim 4 or 5, characterized that at least ten samples, preferably at least a hundred samples or at least a thousand samples analyzed simultaneously become. Method according to one of claims 1 to 6, characterized that the label is a fluorescent label. Method according to claim 7, characterized, that the fluorescent label by means of one or more of the following Marking agent is achieved: Cy3, Cy5, fluorescein, Texas Red, Alexa fluorine dyes and other fluorescent dyes. Process for the preparation of target molecules which exclusively in spatial Close to respective target sequence or within the respective target sequence have at least one mark. A method according to claim 9, characterized in that the labeling of the target molecules by enzymatic end-labeling, reverse transcription, indirect Marking and / or "sandwich" methods take place. Process according to claim 9, characterized records that target molecules are thereby generated by means of a PCR method in which at least one marker provided with a primer is used. Method according to claim 11, characterized in that that at least another one with an identical or another Labeling primed primer is used. Process for the preparation of target molecules which a marker in spatial Close to Having target sequence, wherein the target molecules by means of a PCR method and at least one type of dNTPs are used which are provided with a marking agent, and in which the labeled dNTPs in close proximity to the target sequence and / or be incorporated into the target sequence itself in the target molecule. Process for the preparation of target molecules which a marker in spatial Close to or within the target sequence, in which target molecules by means of a PCR method are generated in which at least one kind dNTPs are used which are provided with a marking agent where the labeled dNTPs are in close proximity to the target sequence and / or incorporated into the target sequence itself, and in which one or more primers are used or used, the one or more provided with the same or further labeling agents is or are. targets for use in a method according to claims 1 to 8, which is a marker or a buildup Markings in close proximity to or within the respective target sequence. targets according to claim 15 prepared by a method according to one of claims 9 to 14. A method according to any one of claims 1 to 8, wherein target molecules are according to Claim 15 or 16 are used. Kit for providing target molecules for performing a Method according to one of the claims 1 to 8, comprising: - primer for the generation of target molecules, the exclusively a label adjacent to or within the target sequence have, and - possibly Buffers and other auxiliary substances. Set of catcher molecules, to execution A method according to any one of claims 1 to 8, comprising: - a predetermined Including number of different capture molecules each different catcher sequences, each for forming ligand bonds with target sequences of target molecules are formed such that they are complementary to the respective sections are the target sequences that are in spatial proximity to a marker or an accumulation of markings. Set according to claim 19, characterized in that the marking or accumulation of labels less than 100 bases, preferably less than 50 or 30 bases apart from the target sequence or themselves located within the target sequence. Set according to claim 19 or 20, characterized that the capture molecules oligonucleotides are. Kit according to one of Claims 19 to 21, characterized that he has at least 10, 30, 50, 100, 1,000 or 5,000 different capture sequences having. Set according to one of Claims 19 to 22, characterized that the catcher sequences constituents are the probes of a DNA array. Method for determining catcher sequences each for Forming ligand bonds with target sequences of target molecules so determined be that they are complementary to the respective sections of the target sequences, which are in spatial Close to a mark or an accumulation of markings. Method according to Claim 24, characterized that the catcher sequences be determined so that the marking or accumulation of Labels less than 100 bases, preferably less than 60, 50, 30, and 20 bases, respectively, from the target sequence located within the target sequence. Method according to claim 24 or 25, characterized that the catcher sequences means of a computer program.