DE10004659A1 - Synthesizing carrier bound arrays of oligomers, useful for performing parallel polymerase chain reactions, e.g. for gene expression analysis, includes reversal of orientation - Google Patents

Abstract

Synthesis of a carrier-bound array (1) of oligomers (2), particularly oligo(ribo)nucleotides, with free ends (A), particularly 3'-hydroxy, and with (i) ends (B), particularly 5'-hydroxy, temporarily protected and (ii) sidechain reactive groups permanently protected. After combinatorial synthesis of (2) on the carrier (9), the temporary protecting group (4) at end (B) is removed and subsequently (but before removing sidechain protecting groups), the free ends (B) are crosslinked by an agent (7). The covalent bond (8) between end (A) and (9) is cleaved for at least a portion (preferably a major portion) of (2) to generate free ends (A), and then a portion (preferably a major portion) of (2) are covalently bonded to (9) through (7). Independent claims are also included for the following: (1) similar method (M1) involving two rounds of combinatorial synthesis; (2) solid-phase PCR (polymerase chain reaction) in which template DNA is hybridized to primers synthesized on a carrier by the new methods; (3) device (I) for analyzing an array of (2); (4) method (M2) of analysis using (I); (5) carrier (II) with at least one array of (2) with free 3'-hydroxy ends for parallel analysis of PCR; and (6) method (M3) for synthesis of carrier-bound arrays of oligomer pairs.

Description

The invention relates to methods and devices for synthesis and Analysis of carrier-bound arrays of oligomers, in particular of primer pairs for PCR (polymerase chain reaction - Polymerase chain reaction), and carriers with oligomers. in the In particular, the invention relates to methods and devices for parallel synthesis of complex oligomer, especially oligo nucleotide libraries.

Here, the term "oligomer" or "oligonucleotide" denotes tid library "the entirety of many different, defi oligomers, peptides bound to supported sites, -nucleotides or -ribonucleotides, the different Oligomers or - (ribo) nucleotides should be arranged as compact as possible should. Such molecular libraries are created in particular by the combinatorial synthesis of a limited number of monomers.

The term "complex" refers to molecule libraries with more than 10 ² different representatives, but in particular molecule libraries with more than 10 ⁴ different representatives.

The invention is intended in particular with a synthesis of primer pairs allow free 3'-OH end for a subsequent PCR  Analysis are suitable and as a couple at a defined location are created on a carrier by combinatorial synthesis.

The previously known solid phase synthesis methods for synthesis of oligonucleotides and the combinatorial synthesis of oligo nucleotide arrays anchor the growing oligonucleotide with the 3'- OH end on the support, while new monomers after cleavage the temporary protective group can be coupled to the 5'-OH end. After the synthesis, all protective groups are split off. At In this last step, the way of anchoring decides Carrier, whether the oligonucleotide with its 3'-OH end on the carrier remains bound, or whether it passes into the soluble fraction. The Synthesis of two different defined oligonucleotide arrays at a defined location in each case has not yet been described.

However, it would be advantageous if defined primer pairs with each free 3'-OH ends could be fixed at a defined location, because z. B. thousands of PCR reactions very easily in parallel be performed.

However, the previous procedures stand in the way, because it is in usually only one defined oligonucleotide per defined location synthesized and the synthesized oligonucleotides bind with their 3'OH end to the carrier.  

Proceeding from this, the invention is based, verbes the task Serte methods and devices for the synthesis or analysis of Oligomers on a support, in particular primer pairs for the PCR (polymerase chain reaction - polymerase chain reaction), and to provide improved carriers with oligomers purified in situ.

The object is achieved by methods and devices with the Features of the independent claims. Advantageous or embodiments are the subject of the dependent claims.

Further details and advantages of the invention result from the following description of some examples in connection with the Drawing. They each show schematically

Fig. 1, I. cleaving off the temporary protective groups 4 at the 5 'OH end of the oligonucleotide 5 2 according to the combinatorial synthesis of a first array of 1 to be agreed locations 10 on the support 9 bound oligonucleotides 2,

Fig. 1, II., The formation of cross-links 7 between the now free 5'-OH ends of the oligonucleotides 5 2,

Fig. 2, III. the splitting off of a second non-permanent protective group 20 which differs from the first temporary protective group 4 on the support 9 , as a result of which a reactive group 22 is formed on the support,

Fig. 2, the structure of a second array of locally precisely defi ned oligonucleotides 2 to the reactive group 22 by means of combinatorial synthesis IV.

Fig. 3, V the formation of cross-links 7 between the free 5'-OH ends 5, each defined precise positions of the second array of spatially well-defined oligonucleotides 2, is produced whereby an array of 21 primer pairs of defined sequence,

Fig. 3, VI. the permanent protective groups 6 are split off, the selection of suitable “handles” 8 ensuring that the majority of the oligonucleotides 2 with a free 3′-OH end 3 are present,

Fig. 4, the hybridization of template DNA 23 at precisely defined locations 10 to a complementary primer 2 VII. Wherein a DNA polymerase starting with the primer 2 synthesized by 3'OH- end 3 of the strand 26 and a controlled heater block 11, the Hybridization temperature, the temperature for the DNA polymerization and the temperature for melting the DNA double strand,

Fig. 4, VIII. Hybridizing the on-the 3'OH end 3 of the primer 2 synthesized DNA single strand 26 after melting of the DNA double strand to the complementary strand primer 24,

Fig. 4, IX. the formation of a double-stranded PCR product 27 after renewed DNA polymerization,

Fig. 4, X. the installation or incorporating a serving for the detection of the PCR product 27 fluorescence dye 25 in the precisely defined positions resulting PCR product 27, which can be propagated by further cycles of PCR,

Fig. 5. I. & II. A process for the synthesis of a carrier-bound array 1 of oligonucleotides 2 with free 3'OH ends 3 , wherein ( Fig. 5, I.) after the combinatorial synthesis the temporary protective group 4 from 5'OH end 5 is split off and then, before the permanent protective groups 6 are split off, the free 5'OH ends 5 are crosslinked, whereupon ( FIG. 5, II.) The covalent bond 8 via the 3'OH end of the synthesized oligonucleotides on the carrier 9 is split off in part, preferably in a predominant part of the synthesized oligonucleotides or oligoribonucleotides 2 , so that free 3'OH ends 3 are formed and a part after the splitting off of these 3'OH ends 3 from the carrier 9 , preferably a predominant part of the synthesized oligonucleotides or oligoribonucleotides 2 can bind covalently to the support 9 due to the cross-links 7 at the 5'OH end 5 ,

Fig. 6 shows a device for parallel analysis of PCR reactions, in which the metallic heating block 11 is milled planar and a fluorescent light filter 18 is provided between a detection unit 17 and an array 1 of oligonucleotides 2 , which fades out excitation light 14 , but emits fluorescent light 19 lets happen.

In order to allow a clear language in the specification 1, the FIG. To FIG. 6, only of oligonucleotides (2) the speech. However, the description also applies to other oligomers ( 2 ), as can already be seen from the labeling table. There are many combinatorial syntheses where the different monomers each have two distinguishable ends A ( 3 ) and B ( 5 ). With nucleotides these are the 3'OH end and the 5'OH end, with amino acids the COOH group and the NH ₂ group, with other monomers there are other groups. The invention relates to all of these different combinatorial syntheses.

At this point it should be pointed out that the invention not only enables the synthesis of oligonucleotides ( 2 ) with a free 3'OH end ( 3 ), but also the in situ purification of carrier-bound oligomers ( 2 ). The longer an oligomer in the combinatorial synthesis is, the more carrier-bound synthesis artifacts (1 not shown in FIG. To FIG. 6) formed, which can make up the predominant part by far of the synthesized oligomers. This is annoying in very many applications, because one would like to attribute a binding or another observable effect to the defined oligomer and not to an undefined artifact. Most of these synthesis artefacts result from the fact that after the temporary protective groups ( 4 ) have been split off, not all reactive groups react with a new monomer. For this reason, these are normally "captured" before a new coupling cycle begins with the cleavage of the temporary protective group ( 4 ). By "capping" the synthesis artifacts lose their reactive group, which means that they cannot be cross-linked ( 7 ). Finally, after the crosslinking ( 7 ) a part (preferably a very large part) of the ends A ( 3 ) is split off from the support ( FIG. 3, VI), the same part of synthesis artifacts is split off. Since these are not cross-linked ( 7 ), they no longer bind to the carrier ( 9 ) and can be washed away.

To the synthesis of fixing on a carrier defined primer pairs to reach each free 3'-OH ends at a defined location, the combinatorial syntheses known in the prior art are amended as follows (see Fig. 1, Fig. 2 and Fig. 3):

As is schematically indicated in FIGS. 1, I and II, in a first step after the combinatorial synthesis of a first array 1 of oligonucleotides 2 bound to the carrier 9 at specific, precisely defined locations 10, the temporary protective groups 4 (an example of such a protective group is split off 4 ', 4'-dimethoxytrityl chloride (DMTr)) at the 5'-OH end 5 of the oligonucleotides 2 and then the now free 5'-OH ends 5 are cross-linked via cross-links 7 . This is done in a manner known per se.

As shown schematically in FIGS. 1, II, and Fig. 2, III, is at closing in a second step a different from said first temporary protective group 4 second non-permanent protective group 20 (eg. B. 2-Acetyl-5, 5-dimethyl-1, 3-cyclohexanedione, (Dde)) split off on the support 9 . This also happens in a manner known per se. The second non-permanent protective group 20 can be introduced during the above-mentioned crosslinking of the free 5'-OH ends, or else it could have been applied to the support 9 prior to the synthesis of the first array 1 of oligonucleotides 2 bound at certain locations 10 .

In a third, in the Fig. 2, III & IV and V step schematically illustrated Fig. 3, produced by the cleavage of the second non-permanent protection group 20 on the support 9 is a reactive group 22 (eg. As a Hydroxylguppe or Amino group) on which, with the aid of known combinatorial syntheses, a second array of precisely defined oligonucleotides 2 can be built up, the free 5'-OH ends 5 of which are crosslinked as described above (crosslinking 7 ). This creates an array of primer pairs 21 of a defined sequence, each of which is defined in a precise location.

Subsequently, as indicated in FIG. 3, VI, the permanent protective groups 6 are split off in a known manner in a fourth step, the selection of suitable “handles” 8 ensuring that the majority of the oligonucleotides 2 are now free 3 ' -OH- end 3 are present. The further anchoring 8 of the oligonucleotides 2 is preferably achieved by incomplete splitting off of the handles 8 mentioned , e.g. B. by derivatizing the carrier 9 with a mixture of handles 8 , part of which is split off under the selected conditions, while a preferably smaller proportion of the handles 8 remains covalently connected to the carrier 9 under the selected conditions.

This method according to the invention has the advantage, among other things,

• - That instead of an array of individual, precisely defined nated oligonucleotides an array of defined oligonucleotide Mating arises,
• - that the oligonucleotides instead of a free 5'-OH end free 3'- Have OH ends and thus a template-dependent potential Represent a substrate for DNA polymerases,  
• - That an in situ purification of the defined oligonucleotides takes place.

In particular, the latter cleaning up of only exemplary and The oligonucleotides mentioned without limitation have large in practice Advantages and can also be used without problems instead of with oligonucleotides other oligomers, such as. B. RNA or peptides applied become. So the majority of the previously known methods Synthesis artifacts no free 5'-OH end and is thus the one above described first step not cross-linked. With the described fourth step, however, is cross-cut along with the 5'OH end cross-linked oligonucleotides also make up the majority of the synthesis artifacts split off and can then be washed away from the wearer become.

The primer pairs 21 described can be used for a precisely defined "solid phase PCR". Compared to conventional PCR methods, thousands of PCR reactions can be carried out very easily in one reaction vessel. Fig. 4 VII. Shows how hybridized template DNA 23 at precisely defined locations 10 to a complementary primer 2. A DNA polymerase synthesizes the counter strand 26 starting from the 3'OH end 3 of the primer 2 . A controllable heating block 11 provides the heat required for hybridization, for DNA polymerization and for melting the DNA double strand.

In FIG. 4, VIII. As shown, as the shown in Fig. 4, VII to the 3'OH end 3 of the single-stranded DNA hybridized to the complementary strand primer 24 after melting of DNA double strand primer 2 synthesized 26th

Fig. 4, IX. illustrates the formation of a double-stranded PCR product 27 after renewed DNA polymerization.

A fluorescent dye 25 , which is used to detect the precisely defined PCR product 27 that can be multiplied by further PCR cycles, can be installed or incorporated as indicated in FIG. 4.

A method for the synthesis of a carrier-bound array 1 of oligonucleotides 2 with free 3'OH ends 3 is described below with reference to FIG. 5.

As shown in FIG. 5, I., after the combinatorial synthesis has taken place, the temporary protective group 4 is cleaved from the 5'OH end 5 . Subsequently, the free 5'OH ends 5 are connected by crosslinking 7 before the permanent protective groups 6 are split off.

Subsequently, as shown in FIG. 5, II., The covalent bond 8 is cleaved via the 3'OH end of the synthesized oligonucleotides to the carrier 9 in part, preferably in the majority, of the synthesized oligonucleotides or oligoribonucleotides 2 , so that free 3rd 'OH ends 3 arise. After cleavage of these 3'OH ends 3 from the carrier 9 , part, preferably a predominant part, of the synthesized oligo nucleotides or oligoribonucleotides 2 binds covalently 8 to the carrier 9 due to the above-mentioned crosslinking 7 at the 5'OH end 5 .

FIG. 6 schematically shows a device for the parallel analysis of PCR reactions, in which the metallic heating block 11 present in most commercially available PCR machines has been milled planar, so that a close contact 12 with a planar array 1 of oligonucleotides 2 with free ones 3'OH ends 3 can arise. When using such a device, an array 1 of oligonucleotides 2 is covered with an exchangeable, transparent, in particular also transparent to UV light planar film or disk 13 , which can be fixed on the array 1 , so that evaporation of the reaction buffer is avoided .

In order to enable parallel detection of the double-stranded DNA 25 resulting from the PCR reactions, the named array 1 is irradiated with oligonucleotides 2 with excitation light 14 , which is particularly suitable for the double-stranded DNA 25 formed with the ge Associate fluorescent dye 15 . The fluorescent dye 15 is excited by an excitation light source 16 mounted above the array 1 of oligonucleotides 2 .

A fluorescent light filter 18 is mounted between the detection unit 17 , which in a particularly advantageous manner consists of a digital camera, and the aforementioned array 1 of oligonucleotides 2 , which filters out the excitation light 14 but allows the emitted fluorescent light 19 to pass.

The data recorded by the detection unit 17 are transferred to an essentially commercially available computer and subjected to an image analysis there.

Below is an inventive device for parallel Analysis of PCR reactions using an array of Oligonucleotides with 3'OH free ends and the use of a described such device.

The device for parallel analysis of PCR reactions is based on essentially commercially available PCR machines, which are modified as described below in order to enable a particularly advantageous, because very simple analysis of many PCR reactions at the same time:

• a) The one available in most commercially available PCR machines Metallic heating block is milled planar, creating a tight Contact with a planar array of free oligonucleotides 3'OH ends is made possible.  
• b) The array of oligonucleotides mentioned is with a interchangeable, transparent, especially for UV light clear planar film or disc covered on the Array can be fixed so that the evaporation of the Reaction buffer is avoided.
• c) The above is used for the parallel analysis of PCR reactions Array of oligonucleotides irradiated with excitation light, esp especially with UV light, which is particularly suitable for a fluorescent dye, especially ethidium bromide to stimulate.
• d) The excitation of the fluorescent dye is done by one mounted above said array of oligonucleotides Excitation light source.
• e) Between the detection unit, which is particularly advantageous Way consists of a digital camera and the array mentioned A fluorescent light filter is mounted on oligonucleotides the excitation light fades out, but the emitted fluorescent light lets happen.
• f) The data recorded by the said detection unit transferred to an essentially commercially available computer and subjected to an image analysis there.

Arrays of oligonucleotides with free 3'OH ends produced in accordance with the invention for the parallel analysis of PCR reactions and their possible uses are described below. Materials produced according to the invention include the molecular libraries produced using the methods, materials or devices mentioned, in particular oligonucleotide libraries with free 3'OH ends. Characteristic of these oligonucleotide libraries is

• - that by combinatorial synthesis of a limited number of suitable nucleoside monomers,
• - that they act as a 2-dimensional array on a suitable deriva tized carriers are present, the individual components of the Oligonucleotide library can be assigned precisely (the The carrier is derivatized in a manner known per se Wise),
• - that they have a free 3'OH end that after hybridization of template DNA substrate of template-dependent DNA poly merase is
• - That preferably two defined oligonucleotides per defined Were synthesized on site.

The oligonucleotide libraries mentioned are used for very simple PCR analysis, especially of complex template mixtures. In doing so, reproduce the synthesized on a type of array Primer pairs of suitable sequence regions of preferably one Variety of different genes of pathogens, so that  can be diagnosed at the same time whether an infection with a this pathogen is present or not.

For example, on an array with 10 ⁵ different primer pairs, 1000 infectious diseases with 100 different primer pairs each can be covered, including almost all known human pathogenic virus genomes (e.g. hepatitis A, B, C, HIV, papilloma viruses, rhinos, flu viruses, etc. ), Bacterial genomes (e.g. Helicobacter pylori, Haemophilus influenza, E. coli etc.) and the genomes of various human pathogenic unicellular organisms (e.g. Entamoebae histolitica, Plasmodium, Trypanosomes etc.).

In another type of array, the syntheti reproduced thereon based primer pairs of suitable sequence areas of preferably as many human ESTs (Expressed Sequence Tags) as possible, so that after hybridization of complex cDNA a so-called "Expression Profiling" can be performed. It will be parallel analyzes which and how many mRNAs (and thus those of them derived cDNAs) in a preferably human tissue or a preferably human cell line can be expressed. This type of array is particularly suitable for comparison of several complex cDNAs with each other and thus for the Identification of comparatively up-regulated or down regulated genes.  

With such a type of array, genomic DNA can also be analyzed become. With the help of a quantitative PCR z. B. find out which genome areas are homozygous or are heterozygous deleted, with the aim of these areas in turn one assign genetic disease.

The arrays of primer pairs mentioned can be reused if, after the PCR reaction, the filters are mixed with suitable Restriction endonucleases are digested and then heated and non- carrier-bound DNA is then washed away. The prerequisite is that the 3 'ends of the primers used are one of several suitable recognition sequences for the named Restriction endonucleases included. This procedure is particularly important especially useful when two different complex templates be compared with each other, comparative quantitative Data should be preserved and thus possible variations in the Filter production should be avoided.

A special feature of the arrays mentioned is one so far unmatched sensitivity to analysis because of the measurement principle of these arrays based on the polymerase chain reaction (PCR) compared to the hybridizations used so far is much more sensitive.

The method described above enables the production of a Arrays of oligonucleotides, each with free 3'OH ends. Becomes Hybridized template DNA to such an array of oligonucleotides,  the resulting complex of carrier-bound Oligonucleotide and template DNA as a substrate for DNA-dependent DNA polymerases are used. Instead, with an RNA template an RNA-dependent DNA polymerase can be used. In order to In principle, such arrays can be used for parallel, for example Sequencing complex templates and or for highly parallelized polymerase chain reactions are used. The The reaction conditions required are known to those skilled in the art.

In the case of analysis of highly parallelized Polymerase chain reactions, it is particularly advantageous if how described above instead of one defined oligonucleotide two defined Oligonucleotides can be synthesized in a site-defined manner. It can especially complex for the subsequent parallel sequencing Templates each one of the primers of the pair of primers compared with the other primer in a significantly smaller amount, so that after a few propagation cycles mostly single-stranded DNA is produced. If ddNTPs are added at this stage, this creates a family of single-stranded oligonucleotides which, for. B. a sequence determination through their analysis in the mass spectrometer the increased template DNA.

With the help of a primer pair, the one typical for PCR can be very easily high specificity can be achieved while using a single primer at least with a complex mixture of templates would result in large proportion of artifacts.  

Another important point is the fact that with the above Arrays the primers are fixed to the carrier, because this is the only way the parallel analysis of many PCR reactions simultaneously in one Reaction vessel, since the otherwise resulting primer mixture too would lead to unpredictable amplicons. This design makes it possible it even, the template DNA used after one or a few Wash away the PCR cycles so that the newly created ones Template DNA is only available in carrier-bound form. In order to the proportion of PCR artifacts is further reduced.

Analysis of highly parallelized polymerase chains reactions are conveniently carried out with the above wrote inventive device for parallel analysis of PCR reactions.

The analysis of the individual fluorescent spots that make up the ent speaking local primer pairs can be assigned after the end of the PCR reaction or, in particular advantageously, "online" with the mentioned inventive Device for the parallel analysis of PCR reactions. Also there the individual fluorescent dots will correspond to the corresponding ones assigned to locally defined primer pairs, but this results in Procedure for each location-defined pair of primers PCR reaction so that the PCR reaction of each primer pair can be quantified.  

The fact can be particularly advantageous be exploited that free nucleotides or single-stranded DNA like the oligonucleotide primers mentioned, a much weaker one Fluorescence signal with embedded ethidium bromide result as double-stranded DNA.

There are also other methods known to those skilled in the art Quantification of PCR reactions, also on the installation or the incorporation of fluorochrome into the PCR reaction formed double-stranded DNA based. Last but not least, there are also Possibility to directly follow the change in absorption that occurs at the Conversion of mononucleotides into double-stranded DNA he follows.

The arrays of primer pairs mentioned can be reused if, after the PCR reaction, the filters are mixed with suitable Restriction endonucleases are digested and non-carrier bound DNA is then washed away. Requirement is, that the 3'OH ends of the primers used are one of several suitable recognition sequences for the restrictions mentioned endonucleases included. This procedure is especially then useful when two different complex templates with each other be compared. Such analyzes should, if possible Receive comparative quantitative data and therefore, if possible Variations in filter production can be avoided.  

A particularly advantageous application of the arrays mentioned results with the almost completely automated parallel diagnosis very many different diseases, especially of Infectious diseases with the help of PCR. It can also Diagnostic certainty for the individual diseases increased significantly be by not just one, but many disease-specific Primer pairs are analyzed.

Another particularly advantageous application of the arrays mentioned lies in creating an expression pattern, especially one comparative expression pattern. The mRNA is one Tissue or a cell line in a manner known per se in (highly complex) cDNA, which in turn is used as a template for the arrays mentioned serves. If these arrays are now primer pairs carry the human EST sequences (Expressed Sequence Tag) can reproduce, so in the mentioned mRNA or cDNA present EST sequences site-specific and almost increased quantitatively. In particular, a comparison of tumor tissue with the surrounding normal tissue results in EST sequences that expressed comparatively strongly or weakly in the tumor tissue are. Due to the immense sensitivity of the PCR Technique also available for analysis and weakly expressed genes comparatively tiny amounts of template can be used be, which makes these filters the current state of the art are superior.  

Another particularly advantageous application of the arrays mentioned is the assignment of homozygous or heterozygous deleted Areas related to somatic genetic diseases and Hereditary diseases. This can be done with the help of those described above quantitative PCR can be found. To do this, an array with suitable primer pairs are used, the sequence regions of the Duplicate genes, duplicate the sequence regions of genes that encode the named ESTs.

The following are some examples of the implementation of the invention method or the use of Vorrich invention described:

A) Synthesis of an array of primer pairs, each with free 3'OH Ends on a support using a converted color laser printer

A suitable carrier with free amino groups (or hydroxyl groups) is manufactured using standard methods. If so not already available through the first step, to the free ones Amino groups (or hydroxyl groups) with the help of a person skilled in the art standard synthesis under anhydrous conditions suitable linker synthesized.

This linker preferably consists of Dde-Fmoc-Lys, one of which Amino group by an fmoc protecting group, the other by a  Dde protective group are blocked. The fmoc protecting group is at Cleaved at 25 ° C for 10 minutes with 20% piperidine in DMF, d. H. under Conditions in which the Dde protecting group remains stable. Subsequently, techniques known to those skilled in the art are used or two RNA phosphoramidites activated with the help of tetrazole and coupled to the carrier. This can occur during the coupling reaction a small proportion of less than 5% of the corresponding DNA Phosphoramidites are added during the coupling reaction.

After splitting off the DMTr protector from the 5'OH end of the the carrier coupled ribonucleoside monomers, the carrier with 4 different toner liquids printed, the 4 different Contain phosphoramidite monomers. The phosphoramidites are with Helped by tetrazole activated, coupled to the carrier, uncoupled Monomers washed away and then the DMTr protective group cleaved from the 5'OH end of the growing oligonucleotide. A Repetition of this process leads to a combinatorial Synthesis of oligonucleotides.

The coupling of the activated phosphoramidites (with protective groups) the carrier, the deprotection and the washing steps can be found under the standard conditions known to the person skilled in the art for oligonucleotide synthesis instead.

After synthesizing a first array of oligonucleotides, at the end the DMTr protective group is split off from the 5'OH end  and the free 5'OH groups e.g. B. with cross-linked cellulose acetate and N, N'-dicyclohexylcarbodiimide (DCC) with the person skilled in the art known conditions cross-linked.

The above-mentioned Dde protective group is then at 25 ° C with 2% Cleave hydrazine in DMF for 10 minutes and be as above first wrote one or two RNA phosphoramidites to the Amino acid of the said lysine linker synthesized. You can the RNA phosphoramidites mentioned as phosphoramidite toner particles printed or evenly in coupling buffer over the carrier be distributed. As described above, during the Coupling reaction a small proportion of the corresponding DNA Phosphoramidites are added during the coupling reaction.

Again, as described above, an array of oligonucleotides becomes synthesized and cross-linked at the 5'OH end. Eventually everyone will Protecting groups with ammonia for 45 minutes at 55 ° C to 70 ° C split off, the carrier washed with acetonitrile and dried, so that in the end a carrier with different defined areas arises, each representing a pair of oligonucleotides. in the given example represent the pairs of oligonucleotides Sequences for the duplication of approximately 50,000 different ones human ESTs. In a final step, the 3'OH ends the oligonucleotides are cleaved from the support with RNase or under split alkaline conditions.  

B) Comparison of the gene expression profiles of tumor tissue with normal tissue

From a patient's tumor tissue and the surrounding tissue Normal tissue is mRNA using the technique known to those skilled in the art obtained, unwritten in cDNA and as a complex template used for the hybridization to the array described under A).

The array mentioned is together with the template cDNA in the parallel analysis device described above clamped by PCR reactions. Then become known suitable thennophilic DNA polymerases in a suitable buffer added and the PCR reaction started. With the in the example mentioned device is one for each defined primer pair Progress curve of the PCR reaction created, which enables each of the ESTs defined this curve by means of the primer pairs assign characteristic value.

The values obtained with cDNA from a patient's tumor tissue are obtained and the values obtained with cDNA from the surrounding normal tissues are compared. This leads to the identification of genes in the tumor tissue are over or under-expressed compared to normal tissue. Another application example is the parallel PCR diagnostics from Pathogens.  

Reference list

1

Array

2

Oligomer, especially oligonucleotide

3rd

free end A, in particular

3rd

'OH end

4

first temporary protection group

5

free end B, in particular

5

'OH end

6

permanent protection group

7

Cross-linking of the free ends B, in particular the

5

'OH ends

8th

"Handle" (covalent bond to the carrier)

9

carrier

10th

defined location on the array

11

planar milled heating block of a PCR machine

12th

Contact point between carrier

9

Heating block

11

13

flat film or disk to cover an array

14

Excitation light

15

Fluorescent dye

16

Excitation light source

17th

Detection unit for the emitted fluorescent light

18th

Fluorescent light filter

19th

emitted fluorescent light

20th

second temporary protecting group

21

Array of oligomer pairs, especially primer pairs

22

reactive group

23

Template

24th

Counter strand primer

25th

fluorescent dye associated with double-stranded DANN, RNA or PNA

26

single-stranded counter strand

27

double-stranded PCR product

Claims (20)

1. Process for the synthesis of a carrier-bound array ( 1 ) of oligomers, in particular of oligonucleotides ( 2 ) or of oligori bonucleotides, with free ends A, in particular with free 3'OH ends ( 3 ), with 5'OH at the -End ( 5 ) of the oligomers a temporary protective group ( 4 ) and permanent protective groups ( 6 ) are provided on reactive side groups, characterized in that

• - After the combinatorial synthesis of the oligomers on the support ( 9 ), the temporary protective group ( 4 ) is split off from the end B, in particular from the 5'OH end ( 5 ),
• that the free ends B, in particular the 5'OH ends ( 5 ), are then crosslinked via a crosslinking ( 7 ) before the permanent protective groups ( 6 ) are split off,
• - That the covalent bond ( 8 ) of the synthesized oligomers, in particular the oligonucleotides or oligoribonucleotides, is then cleaved via the end A, in particular the 3'OH end, to the support ( 9 ) in part, preferably in a predominant part, of the synthesized oligomers can, so that free enes A, in particular free 3'OH ends ( 3 ),
• - That even after the ends A, in particular the 3'OH ends ( 3 ) from the support ( 9 ), part, preferably a predominant part, of the synthesized oligomers due to the crosslinking mentioned ( 7 ) at the end B, in particular on the 5th 'OH end ( 5 ) covalently ( 8 ) binds to the support ( 9 ).

2. Process for the synthesis of a carrier-bound array ( 1 ) of oligomers, in particular of oligonucleotides ( 2 ) or of oligonucleotides, with free ends A, in particular with free 3'OH ends ( 3 ), where at the end B, a first temporary protective group ( 4 ) and permanent protective groups ( 6 ) are provided on the 5'OH end ( 5 ) of the oligomers, characterized in that

• - That in a first step after the combinatorial synthesis of a first array ( 1 ) of at certain, precisely defined locations ( 10 ) on the support ( 9 ) bound oligomers ( 2 ), the first temporary protective group ( 4 ) at the end B, in particular is split off at the 5'-OH end ( 5 ) of the oligomers and then the now free enes B, in particular the now free 5'-OH ends ( 5 ), are crosslinked via crosslinking ( 7 ),
• - That in a second step a second temporary protective group ( 20 ) which is different from said first temporary protective group ( 4 ) is split off on the carrier ( 9 ), as a result of which a reactive group ( 22 ) is formed on the carrier ( 9 ),
• - That in a third step on the reactive group ( 22 ) before preferably by means of combinatorial synthesis, a second array of precisely defined oligomers ( 2 ) is built up, the free ends B, in particular the free 5'-OH ends ( 5 ) as in first step are cross-linked,
• - That in a fourth step the permanent protective groups ( 6 ) are split off, the selection of suitable handles ( 8 ) ensuring that the majority of the oligomers with a free end A, in particular with a free 3'-OH end ( 3 ), are present .

3. The method according to claim 1 or 2, characterized in that the further anchoring of the oligomers is achieved by incomplete splitting off of the handles ( 8 ), in particular by derivatizing the carrier ( 9 ) with a mixture of handles ( 8 ), the egg ner part is split off under the selected conditions, while a preferably smaller proportion of the handles ( 8 ) remains covalently connected to the carrier ( 9 ) under the selected conditions. 4. The method according to claim 2 or 3, characterized in that the second temporary protective group ( 20 ) is introduced during the crosslinking of the free 5'-OH ends in the first step or even before the synthesis of the first array ( 1 ) at certain locations ( 10 ) bound oligomers ( 2 ) on the carrier ( 9 ) has been brought up. 5. A process for synthesis of a support-bound arrays (1) from Oli Gomeres, particularly oligonucleotides or oligoribonucleotides having free 3'OH ends (3), wherein at the 5'OH end (5) of the oligomers a temporary protecting group (4 ) and permanent protective groups ( 6 ) are provided on reactive side groups, characterized in that

• - After the combinatorial synthesis has taken place, the temporary protective group ( 4 ) is split off from the 5'OH end ( 5 ),
• - That the free 5'OH ends ( 5 ) are then connected by cross-linking ( 7 ) before the permanent protective groups ( 6 ) are split off,
• - That the covalent bond ( 8 ) is cleaved via the 3'OH end of the synthesized oligomers to the support ( 9 ) in part, preferably in most of the synthesized oligomers, so that free 3'OH ends ( 3 ) arise so that after cleavage of the 3'OH ends from the support, part, preferably a predominant part, of the synthesized oligomers can bind covalently to the support ( 9 ) due to the cross-links ( 7 ) at the 5'OH end ( 5 ).

6. The method according to any one of claims 1 to 5, characterized in that two different oligomers, in particular two different oligonucleotides or oligoribonucleotides are synthesized per defined location ( 10 ). 7. The method according to any one of claims 1 to 6, characterized in that in the array ( 1 ) more than 100 site-defined oligomers per cm ² are bound to the carrier ( 9 ). 8. The method according to any one of claims 1 to 7, characterized in that DANN, RNA or PNA is hybridized to the array ( 1 ). 9. The method according to any one of claims 1 to 8, characterized in that said arrays ( 1 ) are brought into contact with DNA polymerases or RNA polymerases. 10. The method according to any one of claims 1 to 9, characterized in that said arrays ( 1 ) are used for a PCR analysis. 11. Solid phase PCR, wherein template DNA hybridizes at precisely defined locations ( 10 ) to a primer synthesized according to one of claims 1 to 10 on a carrier and by means of a controllable heating block ( 11 ) for hybridization, DNA polymerization and Melting of the DNA double strand is supplied with the required heat. 12. Device for analyzing the arrays ( 1 ) of oligomers synthesized according to claims 1 to 10, characterized in that

• - That a heating block ( 11 ) is provided with a substantially flat contact surface,
• - That means for irradiating excitation light ( 14 ), in particular UV light, are provided on an array located on the heating block,
• - That a detection unit ( 17 ), in particular a CCD array for fluorescent light ( 19 ) emitted by the array of oligomers, is provided and
• - That between the detection unit ( 17 ) and the array to be examined ( 1 ) a fluorescent light filter ( 18 ) is provided, which fades out the excitation light ( 14 ), but allows the emitted fluorescent light ( 19 ) to pass through.

13. A method for analyzing the arrays ( 1 ) of oligomers synthesized according to claims 1 to 10 using a device according to claim 12, characterized in that

• - That an array to be examined is brought into close contact with the heating block ( 11 ),
• - that the array ( 1 ) is covered with a translucent flat film or disc ( 13 ) to avoid evaporation of a reaction buffer,
• that the array ( 1 ) is irradiated with excitation light ( 14 ), in particular with UV light, for parallel analysis of PCR reactions,
• - That the data recorded by the detection unit ( 17 ) is transferred to an essentially commercially available computer and subjected to an image analysis there.

14. A method for analyzing the arrays ( 1 ) of oligomers synthesized according to claims 1 to 10 using a device according to claim 12, characterized in that the arrays ( 1 ) are analyzed with the device mentioned in claim 12. 15. Carrier with at least one array of oligomers with free 3'OH Ends for parallel analysis of PCR reactions, ge indicates that the array according to one of the claims 1 to 10 described method was applied to the carrier. 16. A carrier according to claim 15 with an oligonucleotide library, since characterized in that the oligonucleotide library by the combinatorial synthesis of a limited number of suitable ones Monomers has arisen. 17. A carrier according to claim 15 or 16 with an oligonucleotide library thek, characterized in that the oligonucleotide library as a 2-dimensional array on the derivatised in a suitable manner th carrier is present, the individual components of the oligo nucleotide library are assigned precisely. 18. A carrier according to any one of claims 15 to 17 with an oligonucleo tid library, characterized in that the oligonucleotide library has a free 3'OH end that after hybridization of  Template DNA substrate from template-dependent DNA Polymerases or RNA polymerases. 19. Support according to one of claims 15 to 18 with an oligonucleo tid library, characterized in that in the oligonucleotide library two defined oligonucleotides per defined location syn were theorized. 20. Carrier with at least one array of oligomers that are in one have been subjected to the situ cleaning step, thereby records that the array according to one of claims 1 to 10 described method was applied to the carrier.