DE10027040A1 - Method of making DNA arrays - Google Patents

Abstract

The invention relates to a method for analyzing nucleic acids. A surface is provided with islands of DNA of the same variety, i.e. tertiary nucleic acids; the tertiary nucleic acids are brought into contact with a probe or a mixture of several probes to enable hybridization to occur between the tertiary nucleic acids and the probe or probes; the tertiary nucleic acids and/or probes which are localized at points where a differential hybridization event occurs are separated from other nucleic acids and/or probes where no differential hybridization event is localized.

Description

The method according to the invention is a method for manufacturing position and use of DNA microarrays, primarily in the field of Gene expression analysis and genome analysis. Another subject of Invention is an apparatus for analysis and photochemical processing said microarrays.

Methods for producing DNA arrays are known from the prior art knows. For example, Maier et al. the production of membrane fil ters with a high density of cDNA clones (J. Biotechnol. 1994 June 30; 35 (2-3): 191-203). Schena et al. (Science 1995 Oct 20; 270 (5235): 467-70) the production of cDNA microarrays, in which known cDNAs on a microscopic slides stored and for hybridization with from to under fluorescent-labeled probes originating from searching biological samples be set. An alternative method for microarray production, which on the construction of oligonucleotides on surfaces via photolithography based, McGall et al. (Proc. Natl. Acad. Sci. USA 1996 Nov. 26; 93 (24): 13555-60).

However, one of the major disadvantages in the production of DNA arrays according to the prior art is the fact that the DNA sequences to be applied to the support surface must be known beforehand. This applies both to photo-lithographically produced oligonucleotide arrays and to "dotted" arrays (separate storage of each individual DNA species), for which a normalized and characterized bank of DNA clones must be present. The characterization (in particular sequence verification of the clones to be deposited) of such banks means an enormous effort, which leads to high costs and results in a clear inflexibility of the method. In particular, the use of conventional prior art array technology for molecularly not very well characterized organisms is excluded. This follows from the very high number of genes of multicellular eukaryotic organisms (an estimated 100,000 different genes in mammals), the extreme size of many eukaryotic genomes (approx. 10 ⁹ base pairs in the human genome; approx. 10 ¹⁰ base pairs in the genome of some agronomically significant ones Useful plants) and the very different expression strength and expression localization of the individual genes. The interaction of the above-mentioned factors means that even an almost complete information about the sequence of the transcripts of an organism (and thus access to each individual transcript) is hardly obtainable with the means available today. Only in the case of a few, particularly intensively investigated organisms (in particular humans, mice, Cae norhabditis elegans, Drosophila melanogaster, and Arabidopsis thaliana), extensive knowledge of the various mRNA molecules can be expected in the foreseeable future. The next step, however, is to generate suitable nucleic acid molecules and to place them on a surface in an orderly manner in a form capable of hybridization, if only not a small selection (for example a few hundred to a few thousand) of different nucleic acid species in the form of a Arrays should be arranged.

The known methods for producing DNA arrays therefore have the following disadvantages:

• - very high effort
• - high time requirement
• - Knowledge of the respective mRNA molecules is required
• - Arrays usually only capture small sections of the total of the mRNA.

Therefore, the task was to provide a manufacturing process and use of DNA arrays, which the aforementioned disadvantages of State of the art overcomes.

The object is achieved by a method for nucleic acid analysis, characterized by the following steps

• a) Primer molecules that form at least one pair of primers are on one Immobilized surface;
• b) One consist of at least two different nucleic acid molecules the nucleic acid mixture is thus in contact with the surface from a) brought that hybridization between primer molecules and nucleic acid remolecules can take place;
• c) The immobilized primer molecules are complementary to Ge gene strand extended to form secondary nucleic acids;
• d) The ge not by the immobilization in step a) to the surface bound nucleic acid molecules are removed from the surface;
• e) The secondary nucleic acids are amplified, tertiary nu small acids are obtained;
• f) The tertiary nucleic acids are with a probe or a mixture brought into contact from several probes so that hybridization between between the tertiary nucleic acids and the probe or probes Can be found;
• g) The tertiary nucleic acids and / or probes located in places where a differential hybridization event is located from the other nucleic acids and / or probes located in locations where no differential hybridization event is located, Cut.

Primer molecules are single-stranded nucleic acid molecules with a length of approximately 10 to about 50 nucleotide building blocks and more. It is DNA Molecules, RNA molecules or their analogues that are used for hybridization with a complementary nucleic acid are determined at least over a portion and as a hybrid with the nucleic acid, a substrate for a double strand represent specific polymerase. The polymerase is preferred way around DNA polymerase I, T7 DNA polymerase, the Klenow fragment of DNA polymerase I, to polymerases that are used in PCR, or also a reverse transcriptase.

A primer pair consists of two types of primer molecules that are attached to areas a nucleic acid, the template according to the known Polymerase chain reaction (PCR) bind in opposite directions, which too flank the amplifying target sequence of the nucleic acid. In these areas it is preferably sequence segments that are in the nucleic acid moles cool the nucleic acid mixture are identical. For example, it can be the nucleic acid mixture is a plasmid library. The primer then preferred in the area of the so-called Multiple Cloning Site (MCS) bind, once above and once below the cloning site. Furthermore, the primers could bind to the sequence segments that the linkers ent speak, which, as described below, ligated on both sides to restriction fragments were. The method according to the invention is preferred with only one primer few performed approximately like the method described in US-A 5 641 658, in which only a pair of primers is used. Preferably bind according to the invention the primers of the primer pair or of the primer pairs to sequence regions which are common to all or essentially with almost all nucleic acids of the nucleic acid mixture are identical. It is conceivable, although not preferred for known reasons, it is moreover, to ar with only a single type of primer molecules work.  

At least one of the primers of a pair of primers preferably has a group, which a by exposure to electromagnetic radiation (for example in UV region) has photochemically cleavable bond. This group is so with to connect the primer molecule that that immobilized on a surface Primer molecule by electromagnetic radiation, i.e. photolytically from the Surface can be removed.

The photolytically cleavable group is preferably a group of the general formula II

Here, X or Y is connected or connectable to the surface during the each other radical with the 5'-OH group of the 5'-terminal ribosyl radical or is connected to a base bound to the 5'-terminal ribosyl radical. n and m are natural numbers from 1 to 30.

Venkatesan and Greenberg (J. Org. Chem. 61 (1996), 525-529) also describe the synthesis of photolytically cleavable linker molecules for the oligonucleotide synthesis which, after synthesis, cleaves the oli Allow gonucleotides from the solid support. Such linkers can also be used instead of Grouping of the general formula II can be used, provided that they are from departing from the procedure proposed in Venkatesan and Greenberg se connects to the 5 'end of the oligonucleotides so that an extension of the Oligonucleotides remain possible through a polymerase.

The term surface denotes an accessible surface of a body Plastic, metal, glass, silicon or similar suitable materials. Before  this is preferably flat, in particular flat. The surface can have a swellable layer, for example made of polysaccharides, poly sugars alcohols or swellable silicates.

Immobilization means the formation of interactions with the above written surface, which at 95 ° C and the usual ionic strength and the tem PCR temperatures are stable, d. H. a half-life greater than 1 minute is preferred have more than 5 minutes. It is preferably kova lent bonds that are also cleavable - especially by exposure to electroma electromagnetic radiation and suitable reagents. Prefers the primer molecules are immobilized on the surface via the 5 'termini. Alternatively, immobilization can also take place via one or more nucleotide structures stones that lie between the terms of the relevant primer molecule are unsuccessful gene, although a sequence segment of at least 10 nucleotide components NEN must remain unbound from the 3 'terminus.

Methods are known from the prior art, chemically suitable derivatives bound oligonucleotides to glass surfaces. Are particularly suitable for this purpose, for example, terminal to the 5'- via a polyatomic spacer Primary amino groups (“aminolink”) bound at the end of the oligonucleotide, which can be easily incorporated in the course of oligonucleotide synthesis and can react well with isothiocyanate-modified surfaces. At for example, Guo et al. (Nucleic Acids Res. 22 (1994), 5456-5465) Process to acti glass surfaces with aminosilane and phenylenediisothiocyanate four and then bind 5'-amino-modified oligonucleotides to it. The carbodiimide-mediated bond is particularly 5'-phosphorylated Oligonucleotides on activated polystyrene supports (Rasmussen et al., Anal. Biochem. 198: 138-142 (1991). The use is also commercially available Licher glass carrier, which on the one hand to bind Aminolink-modified Oligonucleotides are activated and on the other hand a structured surface  which have a larger binding capacity compared to a flat surface has capacity (Surmodics, Eden Prairie, Minnesota, USA). Another be known method uses the high affinity of gold for thiol groups to the bin formation of thiol-modified oligonucleotides on gold surfaces (Hegner et al., FEBS Lett. 336 (1993), 452-456).

For example, the nucleic acid mixture in can be a library are nucleic acid molecules that are identical over long distances Show sequence, but in a partial area in the middle of the identical area clearly differentiate. The libraries often consist of, if necessary linearized plasmids, in which various nucleic acid fragments are included were kidneyed. Furthermore, the nucleic acid mixture can be restrictions act fragments, at the intersecting linker molecules of the same sequence left were greeted. The linkers that are at the 5 'end usually differ of the fragments are bound by the linkers attached to the 3 'end of the Fragments are bound. In any case, the interested person is usually sequence section in the nucleic acid molecules of the nucleic acid mixture of two flanking each essentially on all nucleic acid molecules surrounding identical sequence sections.

In step b) the nucleic acids of the nucleic acid mixture are so with the Surface contacted that hybridization between primer molecules and nucleic acid molecules can take place. First, the water fabric backing that is between the primer molecules and between the nucleus have formed acid molecules of the nucleic acid mixture by denaturation (usually by thermal denaturation). Then the Transition to non-denaturing conditions that occur in a particular Speed must be achieved to enable specific hybridization. These processes are known to the person skilled in the art from the polymerase chain reaction.  

The term secondary nucleic acid denotes those nucleic acid molecules which result from complementary extension of primer molecules, whereby the extension is carried out complementarily to the nucleic acid molecules with the primers were hybridized. Because the primer molecules to be extended a surface are immobilized, the result of this extension are also The secondary nucleic acid molecules immobilized on the surface.

The nucleic acids not bound to the surface by immobilization Molecules are removed from the surface in step d). This applies to both extended or non-extended Nu hybridized with primer molecules small acid molecules as well as for the unhybridized nucleic acid mo molecules of the nucleic acid mixture. Usually this removal is done under de naturing conditions (high temperature and / or chaotropic reagents), so that ge also via hydrogen bonds to immobilized molecules bound (hybridized) nucleic acid molecules can be removed. Here it is possible, but not preferred, to remove the aforementioned nucleus acid molecules only after going through one or more amplification cycles to make.

The term tertiary nucleic acids refers to the se functioning as a template secondary nucleic acids as well as those nucleic acid molecules that after the Methods of PCR are formed. It is important that the Ober nucleic acids also removed from the liquid surrounding the surface towards the reaction space. This usually creates real Islands, that is, discrete areas on the surface, the tertiary nucleic acids the same type, that is, identical or complementary nucleic acid moles cool, wear.  

In step (f), the tertiary nucleic acids are incorporated into them by denaturation cell-stranded form transferred to hybridization between the tertiary nu to enable small acids and the probe or probes. Here is before trains, to avoid recombination and renaturation melted Nucleic acid double strands one of the two complementary single essentially remove strands from the surface. Usually happens this in two successive steps, starting with the Amplifika tion present nucleic acid double strands, preferably by using a Re restriction endonuclease to be cut. Then in a second step by denaturing and washing, no longer by covalent bin immobilized single strand fragment from surface and from solution space removed. It is also conceivable for one of two to form a pair of primers Primer molecules by means of a chemically, photochemically or thermally fissionba immobilize their atomic group on the surface and this group in step (f) to split. In any case, in the subsequent denaturation, the full constant counter strand to the still immobilized nucleic acid strand free set and removed. Then the immobilized generated in step (f) are single-stranded nucleic acids under hybridization conditions with min at least one suitably labeled, serving as a probe of nucleic acid mo brought into contact. Particularly suitable as a probe are mRNA or cDNA or nucleic acid mixtures generated from genomic DNA. This Wed creations can contain a uniform marking group. More preferred however, the nucleic acid mixtures contain at least two separable marker groups, which otherwise widen the distinction based on identical molecules from different biological sources.

Within the scope of the invention, a probe is a mixture of at least two ver different nucleic acid species which have one or more detectable markers stations, preferably fluorescent labeling groups or by coupling Fluorescence groups detectable groups. A probe can be in the frame the invention both from one and from several biological samples, ins  special RNA or mRNA or genomic DNA from a specific Ge weave or from a tissue in a certain physiological state, getting produced. A probe is also a mixture of Son the one in the above sense, which however derives from different biological samples were put. In the event that the probe is from different biological samples was prepared, enables sample-specific labeling of the nucleic acid respezies the clear assignment between nucleic acid species and the Her the nucleic acid species concerned comes from a specific biological Sample.

In step (g), the differential Hy bridization events determined. Show differential hybridization events indicates that a type of nucleic acid concentrated on one surface remolecules more hybridized with a probe or with a probe mixture than with another probe or with a different mixture of probes.

Such events can be recognized particularly reliably if, as in Schena et al. (Science 1995 Oct 20; 270 (5235): 467-70) describes a competitive hybridization in which the probe used consists of a mixture of differently fluorescently labeled nucleic acids obtained from different (in particular two) samples. First, the surface on which the hybridization has taken place is scanned, that is to say the locally bound amount of probe is determined (for example by measuring the fluorescence activity). When using a probe which consists of a mixture of different fluorescence-labeled nucleic acids obtained from different (in particular two) samples, scanning can mean the simultaneous or sequential determination of the fluorescence activity at two different excitation and / or emission wavelengths. The spatially resolved fluorescence signals have to be calibrated for each fluorophore. This calibration can be done in different ways. For example, it is possible to work with an internal standard, as described in Schena et al. is described. In this case, a mixture of differently labeled cDNA of different origins would be used as the probe by first transversely transcribing an mRNA of a certain origin together with a known mRNA of known concentration and fluorescence-labeled (e.g. with fluorescein) and the same with the mRNA of the other origin but uses a different fluorescence marker (e.g. Lissamin). If a defined amount of nucleic acid, to which the differently labeled samples of the internal standard bind, is applied to a defined location on the surface, then the local fluorescence signals determined by scanning the surface can be related to the internal standard, as described in Schena et al. described. Alternatively, one can also form the quotient from the local signal intensity with respect to both fluorophores and arithmetically average the number of measurement locations. The selectivity factor obtained in this way

gives approximation indicate the relative sensitivity of the detection of the two fluorophores a and b, if the mRNA samples from different origins are not too low to separate (Sa (x, y) = fluorescence intensity due to the fluorophore a at the measuring point with the coordinates x, y in a matrix from x = 1 to X and y = 1 to Y, Sb (x, y) analog). It often appears appropriate to use signals that are unspecific bond to subtract from the fluorescence signal before the Selek activity factor and all other data can be determined from the raw data. This however, presupposes that the non-specific binding is determined precisely enough leaves, which will not always be the case, so that often on such a Cor rectification is dispensed with.

The selectivity factor _ab can thus be compared to Schena et al. can be calculated using an internal standard or the simplified definition above can be used. A differential hybridization event by definition has occurred when the local quotient of the fluorescence intensities due to fluorophore a and fluorophore b is greater than g. _from or less than 1 / g. _ab , where g is greater than 1.5, preferably greater than 2, especially greater than three.

A differential hybridization event has a quotient Sa / Sb at location x, y in a matrix from x = 1 to X and y = 1 to Y, which is characterized by one of the following inequalities:

The resolution, i.e. the values X and Y of the x, y matrix are determined by the Resolution of the scanning process limited. If a CCD chip is used, it can X.Y mean the resolution of the chip.

It is also possible to use two or more Fluoro at the same time phore, each of which is coupled to an mRNA of a specific origin dispense. In this case one would have to use two (or more) probes, the carry the same fluorophore, but they target mRNA of different origins decline. Then a first hybridization with probe a carry out the first hybridization result by spatially resolved fluorescence Detect measurement, remove hybridized probe a, a second hybridization Perform with probe b, detect the second hybridization result (and possibly further cycles consisting of the removal of a hybridi based probe, hybridization of a further probe and detection of the hybridi perform the result of the hybridization) and the hybridization results obtained compare. The equation above and the inequalities remain applicable, however the indices a and b then mean the respective ones Measuring runs when using probes a and b, which differed on samples origin.  

To decide whether a location x, y is part of an authentic differential hybrid event, the quotients Sa (x, y) / Sb (x, y) of neighboring locations should be compared. Areas that have a differential hybridization event usually have clonal islands on the surface and ha ben thus a substantially round shape and a diameter greater than 0.1 µm, preferably greater than 0.75 µm or 1.0 µm on the surface, especially 1.2-2.0 µm. This filter criterion largely prevents artifacts (see example 7).

The areas where differential hybridization events have occurred ben, are treated selectively such that the molecules immobilized there at least partially detached from the surface. Preferably happens this through locally limited exposure to electromagnetic radiation, in particular their UV radiation or radiation in the visible range, which is able To bring about photochemical cleavage of a photolabile group, which with ei nem primer molecule of a primer pair is connected. It is important that le only those areas are exposed to electromagnetic radiation, a detachment and recovery of the nucleic acid molecules wishes. After selective detachment of the nucleic acid molecules attached to a differential hybridization event are involved, these are carried by the carrier washed.

For analysis of the detached from the surface in (g) as well as in washing solution Nucleic acid molecules are usually first duplicated be necessary, which is preferably by means of in vitro amplification or via toilet nation or a combination thereof. The more to be done Analysis steps are based on the procedures familiar to a person skilled in the art according to the question being pursued, but usually become a se include the nucleic acids obtained. Often, the ones obtained  Nucleic acids also for searching genomic or cDNA libraries used. When using the method according to the invention Sions analysis is usually a check of the sequences obtained using a Quantification method, for example Northern hybridization or quanti tative PCR. Furthermore, the results can be queried electronically Databases, for example sequence databases, used or in turn in Databases are fed.

In another embodiment of step (g), the non-differential Hybridization events involved and / or the nucleic acid molecules involved in them hybridized probe molecules changed so that they are ready for detachment and / or later reproduction is no longer available, i.e. removed or in to be activated.

Inactivation

The non-differential hybridisies are preferred events involving nucleic acid molecules with electromagnetic Treated radiation of suitable wavelength and a crosslinking reagent, so that it cross-links to the double strands created by hybridization Photo reactions are coming. For example, Spielmann et al. (Proc. Natl. Acad. Sci. USA 89 (1992), 4514-8) the covalent linkage with one another hy bridized DNA strands by intercalation of 4'-hydroxymethyl-4,5,8- trimethylpsoralen with subsequent irradiation at 366 nm. Furthermore, also a networking agent can be dispensed with by using the corresponding nu Small acid molecules and probes irreversible due to electromagnetic effects sibel cross-linked (thymidign dimer formation). Another possibility is that irreversible connection of the corresponding nucleic acid molecules and probes with the surface by local heating.

distance

Of course, in step (g), the first cannot differential hybridization events involved nucleic acid molecules and Probes are removed. This can e.g. B. by the cleavage of photolabile groups in the middle of the linker, which is the extended primer molecules, ie  connects the nucleic acid molecules to the surface. For example, a Construct as specified in formula II can be used.

The nucleic acid moles involved in differential hybridization events The cooler and probes are then removed in a second step, which is no longer must be selective. This nucleic acid mo released in a second step The tubes and probes are analyzed as described above.

Incidentally, the use of LCM (laser captu right microdissection, Emmert-Buck et al., Science 274 (1996), 998-1001) on Iso lation of those nucleic acid islands on which differential hybridization events have taken place, provided that the waiter related to immobilization surface has suitable properties for this. For the application of LCM in For the purposes of the method according to the invention, it would be necessary to have a surface to make available, on the one hand, against the during amplification and hybridization prevailing conditions, on the other hand after contact with thermally softened and again cooled polyethylene vinyl can be removed locally with the acetate transfer film together with this from the carrier. Here for example, one could be chemically suitable for binding nucleic acids modified (Rasmussen et al.) polystyrene film on a plastic or glass be pressed on. To ensure that the transfer between Transferfo lie and and polystyrene film enclosed nucleic acids accessible to ma Chen, the polystyrene film with suitable solvents, for example Acetone to be dissolved.

Areas of application of the method according to the invention are in particular all those questions in which two or more nucleic acid mixtures gene should then be examined whether individual nucleic acid species in the Mixtures in different frequencies are included, and this species representing secondary and tertiary nucleic acids to be isolated.  

This is the case, for example, in the expression analysis, in which the Zu composition of different cDNA preparations is compared and the on is the identification of those cDNA species that are in their Distinguish frequency between preparations. Another application is the comparison of mixtures of DNA obtained from genomic DNA Fragments, whereby often certain fragments (for example by "double restriction digestion "with two different restriction endonucleases are only present in a part of the examined preparations. The like fragments, so far mostly about the known RFLP (restriction question length polymorphism) method (Kiko et al., Mol. Gen. Genet. 172 (1979), 303-12) are suitable, for example, for genomic Mapping and analysis of SNPs (single nucleotide polymorphisms) (Palmatier et al., Biol. Psychiatry 46 (1999), 557-67). Another application area which plays an important role especially in plant breeding, consists in the study of genomic fragments on one side of a recognition site for a particular restriction endonuclease and on its flanked on the other side by a sequence originating from a transposon (Arbuckle et al., WO 99/41415). Since the insertion or excision of Knowing that transposons can influence the properties of an organism Such processes and their exact location in the genome, for example for breeding purposes of great interest.

For example, if the method is used for the expression analysis of two biological If samples are used, the following steps are often used come: First, aliquots of those obtained from both samples RNAs pooled and converted into double-stranded cDNA, which in turn a Restriction digestion, for example with a frequently cutting enzyme such as MboI, is subjected. After ligation double-stranded linker becomes a feast phase amplification performed. For this purpose, primer molecules attached to the by the sequence introduced by the cDNA primer used or to that by the Linker can bind predetermined sequence, immobilized on a surface.  

One of the primer species is attached via a photolabile covalent bond bound the surface. The other of the primers contains the sequence of one Interface for an enzyme that the cDNA restriction fragments in the Re gel will not cut. This is usually a non-cutting enzyme chosen, the recognition point of the recognition point used above frequently cutting enzyme, for example BamHI, if as frequently cutting enzyme MboI was chosen. After applying a reak tion chamber and an annealing mixture on the surface, the An nealing mix in addition to those required for performing a primer extension Reagents also an aliquot of the cDNA fragments provided with linkers contains, secondary nucleic acids are generated as described above. To Removal of non-immobilized nucleic acids and introduction of amplifiers tion mixture, which is necessary for the implementation of the polymerase chain reaction contains necessary reagents, "islands" by solid phase amplification generated tertiary nucleic acids. In order to hybridize individual to obtain stranded molecules is cut with BamHI and the respective strand no longer covalently immobilized by denaturing and washing away. To identify those islands that expressed differentially Containing genes are further aliquots of the isolated RNAs in the presence of fluorescence-labeled nucleotide analogs, for example Cy3-dUTP or Cy5-dUTP, separately reverse transcribed. Here, the one obtained from a sample cDNA with a label and the cDNA obtained from the other sample with the other mark. The samples are mixed together denatured and under hybridization conditions with the prepared, the se brought into contact secondary and tertiary nucleic acid-bearing surface. After hybridization and washing, those of both types of probes become stable fluorescence signals are detected, and are described as in Example the positions of those islands (in Example 7 in terms of their fluorescence also referred to as "spots") determines which above average or Above average few molecules of one probe type compared to Mo molecules of the other probe type are hybridized. These islands, now the best  based on secondary and tertiary nucleotides hybridized with probe molecules acid molecules are now photolytically detached from the surface and washed off . The washing solution now contains a mixture of cDNA Restriction fragments, which differ in both biological samples represent highly expressed genes. To identify the corresponding genes adorn, the cDNA restriction fragments are reamplified by means of PCR; here primers are used which have the same sequence as before the immobilized primers used for solid phase amplification. Because it will usually be a mixture of different fragments a way to separate. This separation can by cloning the reamplification products, but it is also a Size separation via preparative gel electrophoresis, especially polyacrylamide gel electrophoresis, possible. The separated products are used for identification usually sequenced and the sequence used for database queries. Often it is already clear in this way which of the already described and genes entered in the queried databases to the genes between the under searched samples heard differentially expressed genes. In many cases this leads Database query, however, not for an already characterized gene, but only delivers sequence-identical DNA sections (so-called ESTs, expressed se quence tags), which so far have no function and in particular are not yet full permanent cDNA or a corresponding genomic locus was assigned de. In still other cases, the database query does not lead to any similar or significantly partially identical sequence. In the latter two cases one usually tries to make longer (as complete as possible) cDNA molecules of the respective gene. This is about searching cDNA- Banks are possible, but sometimes browsing becomes more genomic Prefer banks. An alternative is that of Frohman et al. (Proc Natl. Acad Sci USA 85 (1988): 8998-9002) described method of "rapid am plification of cDNA ends ", which is the isolation of complete cDNA molecules allowed by in vitro amplification. Furthermore you become an expression quan Connect the identification of the identified genes. Because no clear assignment  a single one of the numerous simultaneously detached nucleic acid islands into one subsequently identified gene is more possible, this can be done on a specific Islet ratio of signal strengths of both probe fluorophores not obtained to Determination of a regulatory factor of the corresponding gene is used become. Rather, one will look at the techniques familiar to the person skilled in the art such as such as quantitative PCR or Northern blotting to measure the amount of express change in the determination of each identified gene. In any case provides the method if it for comparative expression analysis of two Pro ben is used, a mixture of cDNA fragments between the two Samples represent differentially expressed genes. With the analog Genome comparison, on the other hand, isolates genomic sections, which have a changed number of copies (e.g. "loss of heterozygosity" or also amplification of certain genomic sections in tumor tissue) or also occurs only in a part of the genomes examined (e.g. insertions or Deletions or fragments that represent genomic rearrangements).

In a further embodiment of the method according to the invention, the following steps are carried out after step e):

• a) The surface from step e) of the method already described, which he most surface, is brought into contact with a second surface which primer molecules, which form at least one primer pair, immoblili are under conditions in which the primer molecules of the two th surface with the tertiary nucleic acids of the first surface hy can bridify;
• b) The immobilized primer molecules of the second surface are combined complementary to the opposite strand with formation of secondary nucleic acids elongates;
• c) Not by immobilization in step a) to the second surface bound nucleic acid molecules are removed from the second surface distant;  
• d) The secondary nucleic acids are amplified, tertiary nu small acids are obtained;
• e) Steps a) to d) are repeated analogously until the desired number made from prepared surfaces;
• f) The surfaces produced in step e) are steps f) and g) subjected to the procedure already described.

In this embodiment of the method according to the invention, between Step e) and f) of the method already described, a contact replication carried out. In doing so, the two parties involved are drafted as planned as possible brought surfaces so close that the ge on the surfaces hybrid primer molecules or nucleic acid molecules hybridize with each other can and a primer extension is possible. After separating the surfaces amplification takes place in accordance with the polymerase chain reaction to itself known way. This contact replication can be repeated any number of times be, the newly made replica through an untreated surface is exchanged, which only the immobilized primer molecules has. The replicas and the original will be complete or complete a portion of steps f) and g) of the method already described below throw.

To carry out the method according to the invention, a device is further provided with the following components:

• a) a reaction and hybridization chamber in the form of a flow cell,
• b) at least one light source for excitation of those used for marking Fluorophores,
• c) at least one light source for photolysis intended to cleave binding gene,
• d) optionally at least one light source for photochemical or thermal Change of nucleic acid or probe molecules,  
• e) at least one photodetector for measuring the emitted fluorescent beam lung,
• f) optionally a device for scanning the be in the flow cell sensitive surface in the XY direction,
• g) optionally at least one storage vessel for fresh reaction solutions as well as at least one vessel for used reaction solutions and min at least a device for pumping liquids,
• h) if necessary, an electronic computer, which the measured fluorescence stores the intensity of the intensity depending on the position on the surface, if necessary, the data records thus obtained are further processed and given if an automatic selection of the areas of interest on the upper surface and the intended electromagnetic impact Radiation controls.

The reaction and hybridization chamber contains the surface on which hybridization of the single-stranded secondary and tertiary nucleic acids with probe molecules (step (f) of the method according to the invention) and ge if necessary, the amplification takes place beforehand. For example, said Surface simultaneously as a wall, preferably as the upper or lower wall, the Serve reaction and hybridization chamber. The chamber must Observation of the processes taking place on the surface at least partially be made transparent. To establish suitable reaction conditions too the reaction chamber is preferably designed to be temperature-controllable, for example by means of a Peltier element.

The light source for photolysis is preferably used to cleave bonds provided and / or the light source for the photochemical or thermal change of Nucleic acid or probe molecules designed as a laser light source. The one set of pulsed lasers is preferred.

In the context of the invention, a photolytically cleavable compound can be used be, which preferably during the oligonucleotide synthesis in the  Nucleic acid backbone can be incorporated and the subsequent photolyti cleavage of at least part of the oligonucleotide from a surface enables. In a preferred form said photolytically cleavable Ver bond the o-nitrobenzyl basic structure, the compound being cleavable rer linker between the 5'-OH group of a ribose or deoxyribose group and either the 3'-OH group of another ribose group or an atom group that can mediate the immobilization of an oligonucleotide, posi can be used. Compounds which are particularly preferred are those with the reaction conditions prevailing in automatic oligonucleotide synthesis are compatible and therefore directly involved in the automatic synthesis found oligonucleotide can be incorporated.

Such a compound is described by the general formula I

where n and m are independently 1 to 30,
R ^{1 means} dimethoxytrityloxy (DMTO) or a group which enables immobilization to a surface or which permits coupling to an immobilizable compound, and
R ^{2 is} -OP (OC ₂ H ₄ CN) N (CH (CH ₃ ) ₂ ) ₂ or -OP (OCH ₃ ) N (CH (CH ₃ ) ₂ ) ₂ ,

carries, as well as their salts.

Compounds are preferred in which R ^{1 is} dimethoxytrityloxy (DMTO), -NH ₂ , -OH, -OPO ₃ H ₂ , -SH, -NCS, -NCO or

N-succinimidyloxycarbonyl (see above).

Another subject is the use of the verb described as a photochemically cleavable group in the immobilization of Nu small acids. The group can be split by light with a wavelength of 400 nm be.

The drawing serves to explain the invention.

It shows:

Small acid molecules Figure 1 shows the amplification of individual nucleic acid molecules using surface bound primers to islands from each identical amplified Nu.

FIG. 2 shows the preparation of clonal nucleic acid hybridization to the islands;

FIG. 3 shows the hybridization clonal islands of single stranded nucleic acids with labeled nucleic acid molecules;

Figure 4 shows the isolation of nucleic acid molecules involved in a differential hybridization event by photolytic cleavage and re-amplification.

Fig. 5, the evaluation and processing of nucleic acid-islands-bearing surfaces;

6 shows expression analysis of primary providing nucleic acids for use in the Expres.

Fig. 7 manalyse providing primary nucleic acids for use in the Geno;

Fig. 8 is the provision of primary nucleic acids for use in the transpo son-mediated genome analysis;

Fig. 9 shows the result of amplification to a surface.

Fig. 1 shows the amplification of individual nucleic acid molecules by means of surface-bound primers to islands of identical amplified nucleic acid molecules, each in detail
1 the irreversible immobilization of oligonucleotides forming a primer pair,
2 the hybridization of the primary nucleic acids with the surface-bound primers,
3 the formation of secondary nucleic acids by extending the length of the primers,
4 the removal of the non-irreversibly bound primary nucleic acid molecules and amplification of the secondary nucleic acids,
5 islands each with identical, tertiary nucleic acid molecules.

Fig. 2 illustrates the preparation of clonal nucleic acid islets for hybridization. The thickened lines (black or rasterized) symbolize known sequences that flank an unknown sequence (thin black lines).

Ph stands for a photolytically cleavable group which was inserted into the sugar phosphate backbone of one of the amplification primers used in FIG. 1. GGATCC is a recognition site incorporated in the second amplification primer for the restriction endonuclease BamHI. Describes in detail
1 the half-sided dissolving of the amplification products from FIG. 1 by cutting with the restriction endonuclease BamHI,
2 the removal of the nucleic acid strand no longer covalently bound to the surface by denaturation and subsequent washing.

FIG. 3 shows the hybridization of clonal islands from single-stranded nucleic acids with labeled nucleic acid molecules (dashed line).

Fig. 4 describes the isolation of nucleic acid molecules involved in a differential hybridization event by photolytic cleavage and reamplification, in detail
1 the cleavage of the hybridized nucleic acid molecules from the surface by photolysis,
2 denaturing the nucleic acid hybrids split off from the surface and annealing a first amplification primer to the first known flanking sequence of the nucleic acid molecules amplified on the surface,
3 means the extension of the amplification primer hybridized in ( 2 ) and the amplification of the released nucleic acid strand with the addition of a second amplification primer which can hybridize with the second known flanking sequence of the nucleic acid molecules amplified on the surface.

Fig. 5 shows the evaluation and processing of nucleic acid-islands-bearing surfaces, wherein
1 a surface with nucleic acid islands (black dots) formed by amplification with immobilized primers,
2 the conversion of the initially double-stranded nucleic acid molecules into single-stranded molecules by restriction digestion and subsequent denaturation as well as hybridization with two differently labeled nucleic acid samples,
3 the fluorescence image obtained by detection of the first sample (fluorescence signals shown in dotted lines),
4 the fluorescence image obtained by detection of the second sample (fluorescence signals shown hatched),
5 the superimposition of both fluorescence images,
6 the image obtained by superimposing both fluorescence images (signals of equal strength in both channels: white; stronger signal in channel 1: dotted; stronger signal in channel 2: hatched),
7 the isolation of nucleic acid molecules which are involved in a differential hybridization event by photolytic detachment from the support and reamplification
shows.

Fig. 6 shows the provision of primary nucleic acids for use in expression analysis, in detail
1 the cDNA synthesis with biotinylated primer and subsequently binding of the double-stranded cDNA to a streptavidin-coated surface,
2 the restriction step with a first restriction endonuclease (RE1), washing away of the released fragments and second restriction step with a second restriction endonuclease (RE2),
3 the ligation of two different linkers,
4 mRNA,
5 double-stranded cDNA immobilized on the surface,
6 shows a cDNA fragment flanked by two different "overhanging" ends,
7 a cDNA fragment flanked by two different linkers (L1, L2)
shows.

Fig. 7 shows the provision of primary nucleic acids for use in genome analysis, in particular
1 a restriction section of genomic DNA with two different restriction endonucleases (RE1, RE2) and the subsequent ligation of different linkers (L1, L2),
2 the amplification of the ligation products with primers which are complementary to each of the linker strands, it being possible to efficiently amplify only those fragments which are flanked by two different primer sequences,
demonstrate.

Fig. 8 shows the provision mediated transposon primary nucleic acids for use in the genome analysis, in detail
1 the section of genomic DNA (thin double line), which contains known transposons (thick dotted double line), with a restriction endonuclease (RE), followed by the ligation of linkers (thick black double lines),
2 the amplification of the ligation products obtained with a primer which is complementary to one of the linker strands (thick black arrow) and with a primer which is complementary to a known region of a transposon (thick dotted arrow)
shows.

Fig. 9 shows the result of amplification on a surface visualized by the incorporation of Cy3-dUTP during the amplification reaction. 60 cycles of amplification were applied; the following settings were selected for detection using the confocal microscope: objective 10 ×, zoom 4 ×, excitation wavelength 568 nm, detection wavelength 600 nm, PMT voltage 860 V, pinhole 1 , offset-16.

The invention is described in more detail by the examples.

example 1 Preparation of nucleic acid molecules

Male Sprague-Dawley rats were administered gavage 50 mg / kg body weight phenobarbital (Sigma-Aldrich Chemie GmbH, Steinheim) dissolved in 500 ul corn oil (Sigma-Aldrich). Control animals received the same amount of corn oil without the addition of phenobarbital. After 4 hours, the animals were decapitated, the livers removed and used for the preparation of RNA according to standard protocols (Ausubel et al., Current Protocols in Molecular Biological (1999), John Wiley & Sons). The RNA from 5 animals treated with phenobarbital or from 5 control animals was pooled, 10 μg of the pooled RNAs were precipitated with ethanol and dissolved in 15.5 μl of DEPC-treated water. 0.5 µl 10 µM cDNA primer CPB28 V (5'-amino-ACC TAC GTG GAT CCT TTT TTT TTT TTT TV-3 '; ARK Scientific GmbH, Darmstadt) was added, denatured for 5 min at 65 ° C and put on ice. The mixture was mixed with 3 μl 100 mM dithiothreitol (Life Technologies GmbH, Karlsruhe), 6 μl 5 × Superscript buffer (Life Technologies), 1.5 μl 10 mM dNTPs, 0.6 μl RNase inhibitor (40 U / pl, Roche Molecular Biochemicals) and 1 µl Superscript II (200 U / µl, Life Technologies) and incubated at 42 ° C for 1 hour for cDNA first strand synthesis. For the second strand synthesis, 48 ul second strand buffer (Ausu bel et al.), 3.6 ul 10 mM dNTPs, 148.8 ul water, 1.2 ul RNaseH (1.5 U / ul, Pro mega Corporation, Madison, Wisconsin / USA) and 6 ul DNA polymerase I (10 U / ul, New England Biolabs GmbH, Schwalbach) added and the reactions incubated at 22 ° C for 2 hours. It was extracted with 100 ul phenol, then with 100 ul chloroform and precipitated with 0.1 vol. Sodium acetate pH 5.2 and 2.5 vol. Ethanol. After centrifugation for 20 min at 15,000 g and washing with 70% ethanol, the pellet was made in a restriction mixture from 15 ul 10x universal buffer (Stratagene GmbH, Heidelberg), 1 ul MboI (Stratagene, 4 U / ul) and 84 µl deionized water dissolved and the reaction incubated at 37 ° C for 1 hour. It was extracted with phenol, then with chloroform and precipitated with ethanol. The pellet was prepared in a ligation mixture from 0.6 μl 10 × ligation buffer (Roche Molecular Biochemicals), 1 μl 10 mM ATP (Roche Molecular Biochemicals), 4 μg linker ML2025 (produced by hybridization of oligonucleotides ML20 [5'-TCA CAT GCT AAG TCT CGC GA-3 '; ARK] and LM25 [5'-GAT CTC GCG AGA CTT AGC ATG TGA C-3'; ARK] in 1 × ligation buffer), 6.9 µl deionized water and 0.5 U T4 DNA- Ligase (Roche Molecular Biochemicals) dissolved and the ligation performed overnight at 16 ° C. The ligation reaction was made up to 50 μl with water, extracted with phenol, then with chloroform and, after addition of 1 μl glycogen (20 mg / ml, Roche Molecular Biochemicals), precipitated with 50 μl 28% polyethylene glycol 8000 (Promega) / 10 mM MgCl ₂ . The pellet was washed with 70% ethanol and taken up in 100 ul water.

Example 2 Coating of surfaces with oligonucleotides

A primer binding solution was prepared, consisting of 19 mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (Sigma-Aldrich), 100 μl of 1 M 1-methylimidazole (Sigma-Aldrich), each 20 μl of 5'-amino-modified primer -Amino- CPB34 V (5'-Amino-TTA TTA ACC TAC GTG GAT CCT TTT TTT TTT TTT TV-3 '; ARK) and Amino-MLISPh20 (5'-Amino-TTT TTT TTT TTT TTT X TCA CAT GCT AAG TCT CGC GA-3 'with

R = 3'-ribosyl; R '= 5'-ribosyl Eurogentec, Herstal, Belgium). 50 µl of this solution were each in NucleoLink Tubes (Nung GmbH & Co KG, Wiesbaden) and overnight at 50 ° C incubated. The primer binding solution was then removed, and the be layered NucleoLink tubes were washed according to the manufacturer's instructions.

Example 3 Amplification on a surface

To generate surface-bound, secondary and tertiary nucleic acids, 1 μl each of the ligation products produced in Example 1 (control group as well as the phenobarbital-treated group) was mixed with 2 μl 50 mM MgCl ₂ solution, 0.5 μl bovine serum albumin (20 mg / ml; Roche Molecular Biochemicals), 2.5 µl dimethyl sulfoxide, 0.5 µl 10 mM dNTPs, 0.5 µl AmpliTaq (5 U / µl; Perkin-Elmer, Forster City, California / USA), 5 µl 10 × PCR buffer II (Perkin-Elmer) and deionized water mixed in a final volume of 50 µl. This annealing mixture was placed in one of the NucleoLink vessels coated with oligonucleotides in Example 2. The vessel was placed in a well of a UNO II thermal cycler (Biometra medical analytics GmbH, Goettingen). The following temperature program was used for the annealing and the subsequent primer extension: denaturation for 60 seconds at 95 ° C, annealing for 5 minutes at 60 ° C, primer extension for 1 minute at 72 ° C. After Primerex tension had taken place, the vessel was rinsed 3 × with deionized water. To remove the non-covalently bound strands, the mixture was filled 3 times with 80 μl of 0.1 × SSC / 0.1% SDS, heated in a thermal cycler at 95 ° C. for 30 seconds each, the solution was discarded and washed with deionized water. Then was filled with 50 ul of an amplification mixture consisting of 2 ul 50 mM MgCl ₂ , 0.5 ul bovine serum albumin (20 mg / ml), 2.5 ul dimethyl sulfoxide, 0.5 ul 10 mM dNTPs, 0.5 ul AmpliTaq and 5 µl 10 × PCR buffer II in deionized water. The reaction vessel was sealed with the supplied adhesive tape, transferred to the thermal cycler and subjected to the following temperature program: de-naturation 20 sec. At 95 ° C, annealing 30 sec. At 60 ° C, extension 60 sec. At 72 ° C, for 60 cycles. After amplification was complete, the seal was removed and the reaction vessel was rinsed 3 times with deionized water.

Example 4 Probe marking

The total RNA obtained in Example 1 was used to isolate poly (A) RNA using (Oligo dT) Dynabeads (Dynal AS, Oslo, Norway) according to the manufacturer's instructions. For primer annealing, 2 μg of the poly (A) RNA thus obtained were made up to 15 μl with 6 μl of oligo (dT) ₂₁ (50 μM; ARK) with DEPC-treated water and heated to 70 ° C. for 5 minutes and cooled on ice. Then 6 ul 5 × first strand buffer (Life Technologies), 1 ul RNase inhibitor (Ro che Molecular Biochemicals), 100 mM dithiothreitol (Life Technologies), 0.6 ul 50 × dNTP-T (each 25 mM dATP, dCTP and dGTP; 10 mM dTTP; Roche Molecular Biochemicals), 2 µl Cy3-dUTP or Cy5-dUTP (1 mM; Amersham-Pharmacia Biotech Europe GmbH, Freiburg), 2 µl Superscript II (200 U / µl; Life Technologies) added . The synthesis of fluorescence-labeled first-strand cDNA was carried out for 2 hours at 42 ° C. in a preheated thermal cycler. For RNA denaturation, 2.5 μl of 1N NaOH were added and the mixture was heated to 65 ° C. for 10 minutes. After cooling on ice, the mixture was neutralized with 6.2 μl of 1M Tris-HCl pH 7.5, diluted to 400 μl with TE buffer pH 8.0 (Ausubel et al.) And on a Microcon 30 column (Millipore GmbH , Eschborn; centrifugation 10 min. At 13,000 rpm) concentrated to 10 µl.

Example 5 Hybridization

To linearize the clonal DNA islands prepared in Example 3, 50 µl of a restriction mixture each consisting of 5 µl 10 × restriction buffer B (Roche), 0.5 µl bovine serum albumin, and 2 µl BamHI (5 U / µl; Roche Molecular Biochemicals) in deionized water, placed in the NucleoLink tubes and incubated for 1.5 h at 37 ° C. In order to remove the strands no longer covalently bound to the surface after the restriction had been carried out, the restriction mixture was removed, the vessels were rinsed with deionized water and then filled with 80 μl of 1.0 × SSC / 0.1% SDS. It was heated in a thermal cycler to 95 ° C. for 60 seconds, the supernatant was discarded and washed with deionized water. For hybridization, 5 μl of Cy-3 and Cy-5-labeled cDNA were mixed with each other and with 50 μl hybridization buffer (0.25 × SSC, 0.02% SDS, 1% N-lauroylsarcosine, 1% blocking reagent (Roche Molecular Biochemi cals), 1 ug of human C _ot 1 DNA (Life Technologies) was added This hybridization solution was placed in the Nucleolink vessels prepared in Example 3 and sealed. It was denatured in a thermocycler for 2 min at 95 ° C. then the temperature was reduced to 50 ° C. and hybridized for 14 hours, the hybridization solution was removed and the tubes were washed 3 × 5 min at room temperature with 2 × SSC / 0.1% SDS, followed by 3 × 5 Min. With 0.2 × SSC / 0.1% SDS at 42 ° C and finally 1 × 5 min. With 0.1 × SSC / 0.1% SDS at 42 ° C.

Example 6 Identification of differentially expressed genes

The hybridized and washed DNA arrays obtained in Example 6 were analyzed on the confocal microscope. For this purpose, the bottoms of the Nucleo link tubes were separated and glued to microscope slides (neoLab Migge Laborbedarf-Vertriebs GmbH, Heidelberg) using Tesa PowerStrips (Beiersdorf AG, Hamburg). A Leica TCS-NT confocal microscope (Leica-Microsystems Heidelberg GmbH, Heidelberg) equipped with ArKr laser and ArUV laser and a UV 10 × 0.4 NA dry lens was used to examine the hybridization results. The arrays were excited in succession at 568 nm and at 647 nm; The emission was detected at 600 nm or at ≧ 665 nm. Using the control software TCS-NT for Windows NT 4.0 (Lei ca), the entire area of the array to be examined was scanned in individual 250 × 250 nm tiles. The zoom setting for this was 4 ×; the photomultiplier voltage was 850 V. The offset values were -16, the pinhole setting was 0.5. Using the TCS Mosaic (Leica) program, the images obtained for each tile were combined to form an overall image of the array. With the help of the QWin program (Leica), the signals were identified in which the signal strength of the two channels differed by at least a factor of 3. The associated coordinates were determined and the respective areas were approached again. After changing the laser, the areas of the array from which the identified "differential" signals originated were irradiated at 10 W output power with a wavelength of 350 nm for 500 milliseconds each. The scan mirror was not moved for this regioselective radiation. The value of the pinhole setting was 3. After the treatment of all selected areas had been completed, the arrays were removed from the slides and, in order to detach the non-covalently bound DNA molecules, in a 2 ml Eppendorf tube (Eppendorf- Netheler-Hinz GmbH, Hamburg) with 50 µl 1.2 × PCR buffer II with 2mM MgCl ₂ . The fragment solution thus obtained was transferred to a PCR reaction vessel and placed on ice with 2.5 U AmpliTaq, 100 μM dNTPs (final concentration) and 0.5 μM CPB28V and ML20 (5'-TCA CAT GCT AAG TCT CGC GA-3 ') ; ARK) (final concentrations) added. A temperature program consisting of 1 min. Initial denaturation at 94 ° C., then 35 cycles of 30 seconds each was used to reamplify the detached fragments. Denaturation at 95 ° C, 30 sec. Annealing at 55 ° C and 90 sec. Elongation at 72 ° C. The product mixture was cleaned and cloned using QiaQuick columns (Qiagen AG, Hilden) (TOPO TA cloning kit, Invitrogen BV, Groningen / Netherlands). Individual clones were used for sequencing the cloned inserts.

Example 7 Algorithm for determining the locations where differential hybridization events have taken place

,
1. Detection of spatial distribution of the intensities in a sufficiently large resolution over the surface from (a) in both color channels simultaneously or separately using a CCD system or a scanning system. Storage of the XY coordinates and the associated intensity measurements in list format.
2. Determine the intensity distribution (histogram) over all pixels separately for both channels.
3. A background value is determined as the average intensity of the pixels which are within the percentile limits to be defined in the lower region of the histogram (for example from 10% -20%). This is determined separately for both channels.
4. Determination of all areas formed from adjacent pixels (hereinafter referred to as spots) whose pixel intensity values are above the background by a fixed factor, according to the following (or modified) algorithm:

Initial conditions

• a) all pixels are classified as unclassified.
• b) Pixels with the same x, y coordinate of the two channels belong together men.

Iteration step

• a) Take the first unclassified pixel (new viewpoint).
• b) If the pixel does not have the specified factor in any channel Exceeds the background value, it is classified as a background class.
• c) If the pixel in one or both channels exceeds the specified factor for its background value, it is classified as a new spot (classification via a unique identifier).
  • 1. Iterative consideration of all pixels that border on the considered pixel
    • 1. Pixels that are already classified are not considered any further.
    • 2. Pixels that do not exceed the specified factor for their background value in any channel are classified as background class.
    • 3. Pixels that exceed the specified factor for its background value in one or both channels are classified as belonging to the spot.
    • 4. For each pixel newly classified in (cc3), the procedure from (c1) is repeated with the newly classified pixel as the viewing point (recursive method).
  • 2. These steps are only finished when no more pixels can be classified than belong to the spot.
• d) Continue with a) until all pixels are classified.

5. For all spots determined, the following sizes are determined taking into account all pixels classified as belonging and output in a list file:

• a) Average intensity separately for both channels
• b) Standard deviation of the intensities separately for both channels
• c) Center of the pixel area in the defined x, y coordinate system
• d) Intensities from (a) minus the determined background value separately for both channels
• e) Quotient of the two results from (d).

6. To combat artifacts, only spots that meet a set of the filter criteria listed below are considered below. For example, it makes sense to neglect spots with a diameter smaller than 1 µm.

• a) A certain number of pixels is exceeded (spot size)
• b) The number of pixels is not exceeded
• c) A proportional range of the spot to be determined is not violated (length / width, where length = x _max -x _min and width = y _max -y _min )
• d) A criterion for describing the shape of the pixel area is fulfilled (e.g. to Approximately round spot judged by the average distance to the Center from (5c) in relation to the number of pixels)
• e) Standard deviation from (5b) does not exceed a fixed in any channel attached importance.

7. Determination of the average quotient for all spots fulfilling the filter criteria from (6), based on the quotients from 5 (e).
8. Division of all quotients considered under (7) by the average quotient from (7) = normalization of the quotients to the mean of all quotients.
9. Determination of all spots whose normalized quotient from (8) deviates from the mean of all quotients by at least a specified factor of n or 1 / n.

Claims (10)

1. A method for nucleic acid analysis, characterized by the following steps:

• a) primer molecules which form at least one pair of primers are immobilized on a surface;
• b) A nucleic acid mixture consisting of at least two different nucleic acid molecules is brought into contact with the surface from a) in such a way that hybridization can take place between primer molecules and nucleic acid molecules;
• c) The immobilized primer molecules are extended to complement the opposite strand to form secondary nucleic acids;
• d) The nucleic acid molecules not bound to the surface by the immobilization in step a) are removed from the surface;
• e) The secondary nucleic acids are amplified, whereby tertiary nucleic acids are obtained;
• f) The tertiary nucleic acids are brought into contact with a probe or a mixture of several probes in such a way that hybridization can take place between the tertiary nucleic acids and the probe or the probes;
• g) The tertiary nucleic acids and / or probes that are located at locations where a differential hybridization event is located are replaced by the other nucleic acids and / or probes that are located at locations where no differential hybridization event is located. Cut.

2. The method according to claim 1, characterized in that after step e) the following steps are carried out:

• a) the surface from step e) according to claim 1, the first surface is brought into contact with a second surface on which primer molecules, which form at least one pair of primers, are immobilized, under conditions in which the primer molecules of the second surface with the can hybridize tertiary nucleic acids of the first surface;
• b) the immobilized primer molecules of the second surface are extended to complement the opposite strand to form secondary nucleic acids;
• c) the nucleic acid molecules not bound to the second surface by the immobilization in step a) are removed from the second surface;
• d) the secondary nucleic acids are amplified, whereby tertiary nucleic acids are obtained;
• e) Steps a) to d) are repeated analogously until the desired number of prepared surfaces has been produced;
• f) the surfaces produced in step e) are subjected to steps f) and g) of claim 1.

3. The method according to claim 1 or 2, characterized in that in step g) of Claim 1 the probes and / or nucleic acids, which is a differential Hybridization event have caused to be detached from the surface. 4. The method according to claim 3, characterized in that the probes and / or Nucleic acids that have caused a differential hybridization event released from the surface by photolytic cleavage of the hybridization partner become. 5. The method according to claim 3, characterized in that the probes and / or Nucleic acids that have caused a differential hybridization event can be detached from the surface by local temperature increase. 6. The method according to claim 1 or 2, characterized in that in step g) of Claim 1 the probes and / or nucleic acids that are not differential Hybridization event, either due to thermal Action or be cross-linked to the surface.   7. Compound of the general formula I
where n and m are independently 1 to 30,
R ¹ denotes dimethoxytrityloxy (DMTO) or a group which enables immobilization to a surface or which permits coupling to an immobilizable compound, and
R ^{2 is} -OP (OC ₂ H ₄ CN) N (CH (CH ₃ ) ₂ ) ₂ or -OP (OCH ₃ ) N (CH (CH ₃ ) ₂ ) ₂ ,
carries, as well as their salts. 8. A compound according to claim 7, characterized in that R ^{1 has} the meaning dimethoxytrityloxy (DMTO), -NH ₂ , -OH, -OPO ₃ H ₂ , -SH, -NCS, -NCO or N-succinimidyloxycarbonyl. 9. Use of the compounds according to claim 7 or 8 as photochemically cleavable Group in the immobilization of nucleic acids. 10. Device for performing a method according to one of claims 1 to 6, comprising

• a) a reaction and hybridization chamber in the form of a flow cell,
• b) at least one light source for exciting fluorophores used for labeling,
• c) at least one light source for photolysis to cleave bonds provided,
• d) optionally at least one light source for the photochemical or thermal change of nucleic acid or probe molecules,
• e) at least one photodetector for measuring the emitted fluorescent radiation,
• f) optionally a device for scanning the surface located in the flow cell in the XY direction,
• g) if appropriate, at least one storage vessel for fresh reaction solutions and at least one vessel for used reaction solutions and at least one device for conveying liquids,
• h) optionally an electronic computer which stores the measured fluorescence intensity as a function of the position on the surface, optionally processes the data records thus obtained and optionally makes an automatic selection of the regions of interest on the surface and controls the action of electromagnetic radiation provided for this purpose.