EP1244782A2 - Method for carrying out the parallel sequencing of a nucleic acid mixture on a surface - Google Patents

Method for carrying out the parallel sequencing of a nucleic acid mixture on a surface

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Publication number
EP1244782A2
EP1244782A2 EP20000990813 EP00990813A EP1244782A2 EP 1244782 A2 EP1244782 A2 EP 1244782A2 EP 20000990813 EP20000990813 EP 20000990813 EP 00990813 A EP00990813 A EP 00990813A EP 1244782 A2 EP1244782 A2 EP 1244782A2
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step
nucleotide
nucleic acids
nucleic acid
characterized
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EP20000990813
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German (de)
French (fr)
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Achim Fischer
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Axaron Bioscience AG
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Axaron Bioscience AG
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869Methods for sequencing
    • C12Q1/6874Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation

Abstract

The invention relates to a method for carrying out the parallel sequencing of at least two different nucleic acids contained in a nucleic acid mixture, whereby: (a) a surface is prepared which comprises islands of nucleic acids of the same type of tertiary nucleic acids; (b) opposite strands of the tertiary nucleic acids GTN are prepared; (c) the GTN are lengthened by one nucleotide, whereby the nucleotide, on the 2'-OH position or on the 3'-OH position, carries a protective group, which prevents an additional lengthening, and the nucleotide carries a molecular group, which enables the identification of the nucleotide; (d) the incorporated nucleotide is identified; (e) the protective group is removed and said molecular group of the incorporated nucleotide is removed or modified, and; (f) step (c) and the following steps are repeated until the desired sequence information has been obtained.

Description

Nerfahren for parallel sequencing of a Νukleinsäuregemisches at a surface

The invention relates to a Nerfahren for solid phase-supported parallel sequencing of at least two different included in a Νukleinsäuregemisch Νuklein- acids.

An important Nerfahren of biological analysis, the sequence analysis of nucleic acids. Here, the accurate base sequence of the DNA of interest, or R A molecules is determined. Knowing these base sequence for example, allows the identification of specific genes or transcripts, so the linked to these genes messenger RNA molecules, the detection of mutations or polymorphisms, or even the identification of organisms or Niren, which is clearly based on specific Νukleinsäuremoleküle reveal. Conventionally, the sequencing of nucleic acids is by the chain termination method (Sanger et al. (1977) PNAS 74, 5463-5467). For this purpose, an enzymatic addition of a single strand is made to the double strand by a hybridized to said single strand "primer" is usually a synthetic Oligonu- kleotid, is extended by the addition of DNA polymerase and nucleotide. A small addition of demolition nucleotide building blocks, which do not permit further extension after their incorporation into the growing strand, leading to Akkumulati- on of sub-tows having a known, fixed by the respective Abbruchnukleotid end. The resulting mixture of different length strands by gel according to size ektrophorese separated. From the resulting band patterns, the nucleotide sequence of the unknown strand can be deduced. A great disadvantage of said method consists in the required instrumental complexity, which limits the achievable throughput of reactions. At least one track on a slab gel or in the use of capillary electrophoresis, at least one capillary is provided the use of labeled with four different fluorophores Abbruchnukleotiden required for each sequencing reaction. The thus resulting expense limited to the most advanced commercially available automated sequencer, the number of parallel processable sequencing to a maximum of 96. A further disadvantage is the limitation of read length ", ie the number of correctly identifiable bases per sequencing, by the resolution of the gel system. A Nerfahren alternative to sequencing, sequence determination via mass spectrometry, is faster and therefore allows

BESTÄTIGUΝGSKOPIE the processing of more samples in the same time is, on the other hand (e.g., 40-50 bases) is limited to relatively small DNA molecules. In yet another sequencing technology, sequencing by hybridization (SBH, Sequencing By Hybridization;.. See Drmanac et al, Science 260 (1993), 1649-1652), Basenfol- be identified gen tiden by the specific hybridization of unknown samples with known Oligonukleo- , Said known for this purpose, oligonucleotides are fixed in a complex arrangement on a support, hybridization with the labeled, nucleic acid to be sequenced is made, and the hybridizing oligonucleotides can be determined. From the information on which oligonucleotides have hybridized with the unknown nucleic acid, and from their sequence, the sequence of the unknown nucleic acid can then be determined. A disadvantage of SBH Nerfahrens is the fact that the optimum hybridization conditions for oligonucleotides can not be exactly predicted and can accordingly not design a large set of oligonucleotides, on the one hand contain all possible at their given length sequence variations and on the other hand exactly require the same hybridization conditions. Thus, it comes through non-specific hybridization to errors in the sequence determination. In addition, the SBH method can not be used for repetitive regions to be sequenced nucleic acids.

In addition to analyzing the level of expression of known genes such as, Northern hybridization, and quantitative PCR is possible by dot blot hybridization methods are also known which enable the de "ovo identification of unknown between different biological samples of differentially expressed genes.

Such a strategy for expression analysis is to quantify discrete seconds quenzeinheiten. This sequence units (expressed sequence tags) are made in so-called. ESTs. Are sufficient numbers of clones from cDNA libraries derived from each other samples to be compared, sequenced identical sequences can each be detected and counted and the resulting relative frequencies of these sequences in the different samples to be compared with each other (see. Lee et al., Proc. Natl. Acad. Sci. USA 92 (1995), 8303-8307). Different relative abundances of a particular sequence indicate differential expression of the corresponding transcript. However, the described method is very expensive since the sequencing of many thousands of clones is already required for the quantification of the more common transcripts. On the other hand, only a short sequence section of about 13 to 20 base pairs in length is required for unique identification of a transcript in general. This fact is the process of "Serial Analysis of Gene Expression" (SAGE) exploited (Velculescu et al, Science 270 (1995), 484-487). Here short sequence segments ( "tags") are concatenated, cloned and the resulting clones are sequenced. With a single sequence reaction can be determined about 20 tags in this way. Nevertheless, this technology is not yet very powerful, because even have to be carried out very many conventional sequencing reactions for quantification of the more common transcripts and analyzed. Due to the high cost a reliable quantification of rare transcripts by SAGE is very difficult.

A further method for sequencing of tags according to the US-A 5,695,934 is, layers with little balls nucleic acid to be sequenced in such a way into account that each ball only receives numerous molecules of a nucleic acid species. For sequencing, the method of the 'stepwise ligation and cleavage "is then used as base in the degraded by an artificial linker of the nucleic acid to be sequenced by using a type IIS restriction enzyme and its sequence is determined. Thus, an observation and recording of possible sequencing process is lent, the balls used are introduced into a flat cuvette, which is only slightly higher than that corresponding to the ball diameter to allow the formation of a single layer. Furthermore, the balls must be in closest packing in the cell, so that there is gelanordnung during the sequencing process either by the necessary exchange of the reaction solution or by vibrations of the instrument to a change in Ku. Although to be carried out many sequencing reactions in a small space in this way, the arrangement in a very narrow cell (a few microns high) has considerable disadvantages, since a uniform filling of the cuvette is difficult to achieve. Another disadvantage is the high equipment expense of the process. So necessary to work with high pressures, for example, so that despite the small cuvette an efficient exchange of the necessary reaction solutions is possible. Yet another drawback is the easy plugging of the cuvette, which is also favored by the necessarily small dimensions of the cuvette.

The known methods for Nukleinsäureseanalytik have one or more of the fol- constricting disadvantages:

- They allow only very limited scope of the parallelized execution of individual sequencing reactions.

- you need relatively large amounts of nucleic acid whose sequence is to be determined. - They are only suitable for sequence determination of short sequence segments and complicated apparatus. It is an object of the invention to provide a method which overcomes the disadvantages of the prior art.

The inventive object is achieved by a method for parallel sequencing of at least two different nucleic acids contained within a nucleic acid mixture, wherein

(A) is a surface provided, comprising islands of nucleic acids each variety, nucleic acids tertiary;

(B) are opposite strands of the tertiary nucleic acids, GTN provided; (C) the GTN be extended by one nucleotide, wherein

- the nucleotide carrying a protective group at the 2'-OH position or at the 3'-OH position, which prevents further extension

- the nucleotide carries a molecular group which enables the identification of the nucleotide; (D) identifying the incorporated nucleotide;

(E) the protective group is removed, and removes the molecule group used for the identification of the incorporated nucleotide or modified, and

(F) Step (c) and subsequent steps be repeated until the desired sequence information has been obtained.

A specific embodiment of the method according to the invention provides the following represents, wherein in step (a)

(Al) is a surface provided, to which at least primer molecules a first primer and a second primer, and optionally a nucleic acid mixture comprising the nucleic acid molecules with which both

can hybridize primers have been irreversibly immobilized, wherein both primers form a primer pair; (A2) the nucleic acid molecules of the nucleic acid mixture are hybridized with one or two primers of the same primer pair; (A3) the irreversibly immobilized primer molecules are extended complementary to the opposite strand to form secondary nucleic acids; (A4) is provided in the surface of a Nukleinäuremolekülen that are not bound by irreversible immobilization to the surface, freed form; (A5), the secondary nucleic acids are amplified to form tertiary nucleic acids. Tertiary nucleic acids in accordance with step (a) can be provided, by starting from a surface to which at least a first primer and a second primer, and optionally a nucleic acid mixture, are irreversibly immobilized comprising the nucleic acid molecules with which both of the primers can hybridize. Both primers form a primer pair can thus bind to strand or strand of nucleic acid molecules. If the nucleic acid molecules of the nucleic acid mixture are already bound to the surface, then the hybridization in step (a2) by merely heating and cooling can be effected. Otherwise, the nucleic acid molecules of the nucleic acid mixture to be brought into contact in step (a2) with the surface. In this connection, reference is also made to WO 00/18957.

A special embodiment of the method according to the invention presents the following, wherein in step (al) a surface to which at least one primer pair forming primer molecules have been irreversibly immobilized, is provided. The individual steps to be carried out can also play as follows in this embodiment:

• primer molecules which form at least a pair of primers are immobilized irreversibly to a surface;

• the nucleic acid molecules are hybridized pair with one or two primers of the same primer, by bringing the nucleic acid mixture with the surface in contact;

• the irreversibly immobilized primer molecules are extended complementary to the opposite strand to form secondary nucleic acids;

• the unbound by irreversible immobilization on the surface Nukleinäu- acid molecules are removed from the surface;

• the secondary nucleic acids are plifiziert am- to form tertiary nucleic acids;

• opposite strands of the tertiary nucleic acids, GTN, are provided;

• the GTN be extended by one nucleotide, whereby the nucleotide bears • at the 2'-OH position or at the 3'-OH position a protective group which prevents further extension

• the nucleotide carries a molecular group which enables the identification of the nucleotide;

• the incorporated nucleotide is identified; • the protecting group is removed and the molecule group of the incorporated nucleotide used for identification is removed or altered, and

• the seventh step and the subsequent steps are repeated until the desired sequence information has been obtained. The nucleic acid mixture of step (a2) may be, for example, to a library, that is to nucleic acid molecules having an identical sequence over long distances, but differ greatly in a section in the middle of the identical portions. Often the libraries consist of optionally linearized Plasmi- the one in which different nucleic acid fragments were cloned to be sequenced later. Further, it may be the nucleic acid mixture to restriction fragments of the same at the cut ends linker molecules sequence were ligated. Here, the linker, the 'bound-end of the fragments of the linkers at the 3' -end to the 5 bound the fragments differ in the rule. In any case, in general, the sequence of interest in the portion of the nucleic acid Nukleinsäuremole- mixture of two flanking substantially small acid molecules with all nucleic each identical sequence sections surrounded cules, of which preferably at least one of the two sequence sections, a self-complementary sequence. in single-stranded form of the sequence section in question has a pronounced tendency to form a so-called shark instruktur.

The primer or the primer molecules in step (al to a3) are single-stranded nucleic acid molecules of a length of about 12 to about 60 nucleotide units and more suitable in the broadest sense for use in the PCR .. These are DNA molecules, RNA molecules or analogs thereof, which are intended for hybridization with a complementary over at least a portion of nucleic acid and represented as a hybrid of the nucleic acid with a substrate for a double strand-specific polymerase. In the polymerase it is preferably DNA polymerase I, T7 DNA polymerase, Klenow fragment of DNA polymerase I to polymerases, which are in the PCR application, or also a reverse transcriptase.

The primer pair in step (a2) represents a set of two primers that bind to regions of a nucleic acid which flank to be amplified target sequence of the nucleic acid and have a "polarity" in relation to the orientation of their binding to the nucleic acid that amplification possible (the 3 'termini face each other). these areas are preferably sequence segments, the acid molecules in the nucleic of the nucleic acid mixture are identical. for example, it may be the nucleic acid mixture to a plasmid library. the primers would then loading vorzugt in the field of so-called multiple cloning site (MCS) bind, once above and once below the cloning site. in addition, the primer can bind to the sequence segments that match the linkers, as described above, to restriction fragments were ligated on both sides. The inventive method is only ei preferably nem Primeφaar performed about method as that described in US-A 5641658 (WO 96/04404) described in which only a Primeφaar is used. According to the invention, the primers of the Primeφaars or Primeφaare to sequence regions which are substantially identical in all or almost all of the nucleic acids bind nucleic acid mixture (so-called conserved regions). The primer of a Primeφaars may have, moreover, the same sequence. This may be advantageous if the conserved regions that flank the sequence to be amplified, having sequences that are complementary to each other.

One of the primers of a Primeφaars may comprise a sequence which allows the formation of an intramolecular double helix nucleic acid (a so-called Haiφinstruktuf), although an existing of at least 13 nucleotide region of the 3 'terminus remains unpaired.

In the surface in step (a, al and a2, a4) is the accessible surface of a Köφers of plastic, metal, glass, silicon, or similar suitable materials. Preferably, this is flat, in particular plane-configured. The surface may be a swellable layer, for example, polysaccharides, polysugar alcohols or swellable silicates.

Irreversible immobilization means the formation of interaction with the above-described surface, which are stable at 95 ° C and the usual ionic strength with the PCR amplifications of the step (a5) in the hour scale. Preferably, these are covalent bonds that can be cleaved. Preferably the advertising primer molecules in step (a) the 5 'termini irreversibly immobilized on the surface. Alternatively, immobilization can also be immobilized via one or more nucleotide tidbausteine, which lie between the respective termini of the primer molecule, although the 3 'terminus must remain unbound a sequence segment of at least 13 nucleotide units calculated from the. the Im- mobilization is preferably carried out by formation of covalent bonds. Here, of course, watch for a corresponding coverage density, which enables contact of at polymethyl rasekettenreaktion involved primer and nucleic acids. Two primers immobilized, the primers should have an average distance to the surface that matches at least in order of magnitude with the maximum length at full extension of the amplified nucleic acid molecules or to but smaller. Here, as in US-A 5,641,658 or WO 96/04404 is to proceed substantially as described. Methods are known to bind chemically suitable derivatized oligonucleotides to glass surfaces from the prior art. Particularly suitable for this purpose, for example, terminal, bonded via a polyatomic spacer to the 5 'end of the oligonucleotide of primary amino groups ( "Aminolink") which in the course of Oligonukleo- can be easily inkoφoriert tidsynthese and can react well with isothiocyanate-modified surfaces. For example, describe Guo et al. To activate (Nucleic Acids Res. 22 (1994) 5456-5465) a method of glass surfaces with aminosilane and Phenylendii- isothiocyanate and then to bind 5 '-aminomodifizierte oligonucleotides thereto. Particularly suitable is the carbodiimide mediated binding Λ 5 -phosphorylierter oligonucleotides to activated polystyrene support (Rasmussen et al., Anal. Biochem 198 (1991), 138-142). Another known method utilizes the high affinity of gold for thiol groups to bind thiol-modified oligonucleotides to gold surfaces (Hegner et al, FEBS Lett 336 (1993), 452-456).

The term secondary nucleic acid in step (a3) ​​refers to those nucleic acid molecules which arise by complementary extension of primer molecules, wherein the extension is complementary to the nucleic acid molecules of step (a2), which were hybridized with the primers.

The surface is in a nucleic acid molecules that are not bound by irreversible immobilization to the surface, freed form provided [Step (a4)]. Provided that the nucleic acid molecules of step (al) in step (al) have already been irreversibly immobilized on the surface, are brought in step (a2) usually no nucleic acid molecules to the surface in contact. Consequently, they must also not be removed in subsequent steps. When in step (a2) the nucleic acid molecules are brought for the purpose of hybridization with the primers having the surface in contact, for example because the nucleic acid molecules (al) have been irreversibly immobilized to the surface not yet in step, this in step (a4) can be obtained by denaturation and wash be removed. It is possible, but not preferred, the removal of the aforementioned nucleic acid molecules only after passing through one or more ammonium plifikationszyklen of step (a5) to carry out.

The term tertiary nucleic acids referred to secondary nucleic acids as well as those nucleic acid molecules from the secondary nucleic acids by the method of polymerase chain reaction in the step (a5) are formed. It is important that the surface and the surface surrounding liquid reaction space are free to be amplified nucleic acids that are not irreversibly immobilized on the surface. By amplification veritable islands usually arise, that is discrete loading rich on the surface, the tertiary nucleic acids of the same kind, that is identical or complementary thereto nucleic acid molecules carry.

In step (b) counter-strands of the tertiary nucleic acids (GTN) may be provided. This can happen, for example, one of three actions listed below:

- On the one hand, at step can (al) Primer molecules or optionally in step (al or a2) nucleic acid molecules (of the nucleic acid mixture) may be used with flanking sequence portions sen the self-complementary regions aufwei- and thus are capable of intramolecular base-pairing which in a so-called Haiφinstruktur expressed (see also Fig. 3: ligation of "masked Haiφins" in the form of double-stranded linker molecules). It is preferred only one primer of a Primeφaars or only a flanking sequence portion of two to form a Haiφinstruktur able to ensure that the , incorporation of nucleotides takes place only at one of two complementary nucleic acid molecules such that an interference of the sequence of signals of both the nucleic acid molecules is excluded.

Then, in the step (a5) tertiary nucleic acids formed are Gigen in einzelsträn- state which is brought about by removal of the conditions of the two strands under denaturing near its 3 'terminus a refolding in the form of a Haiφins on. Preferably, the double-stranded portion of the Haiφins ranges up to and including the last base of the 3 'end, so that said Haiφin can immediately serve as a substrate for a polymerase used for sequencing. This must be ensured by appropriate selection of the sequence of primer molecules or nucleic acid molecules flanking sequence segments.

Secondly GTN can be provided by ligation of oligonucleotides in the form of Haiφins, which are capable of Haiφinbildung and optionally (but not necessarily) in the form of already einge- Haiφins for ligation sets (see also Fig. 2). This can be done so that the tertiary nucleic acids in double-stranded (i.e., undenatured) state cut and separated in this way on one side of the surface. Preferably, this is done by incubation with a restriction endonuclease that in exactly one of originating from one of the two primer sequences (primer sequences) or in a position adjacent to these primer sequences sequences comprises a recognition site. After the restriction a free end of the tertiary nucleic acids in the solution space, which is a protruding end predictable seconds has frequency depending on the restriction endonuclease and to which the oligonucleotide may be hybridized and ligated then projects. this would be particularly suitable is an oligonucleotide which has already formed a Haiφinstruktur, therefore thus partially double is present, and a complementary to the free end of the tertiary nucleic acids overhang sitting loading. In order to ensure that ligation takes place exclusively at the irreversibly immobilized strand of the double strand of the tertiary nucleic acids, the 5 'end of the oligonucleotide carrying a phosphate group, while the 3'-end of the irreversibly immobilized strand and the 5' can-end of the with this hybridized complementary strand having an OH group (see Fig. 2, steps 1 and 2). After ligation of not irreversible immobilized strand of the tertiary nucleic acids is removed under denaturing conditions. Alternatively, as proposed in US-A 5,798,210 (particularly Fig. 7 ibid), a ligation of a refolded to Haiφin oligonucleotide could also be made to the single-stranded present immobilized strand of the tertiary nucleic acids. that often observed inadequate efficiency of the ligation step prior to sequencing can not be compensated by amplification steps as in the first measure is problematic in this second measure. This may have a low signal strength in the subsequent sequencing result.

Thirdly, it is also possible to oligonucleotides that are not for forming a hair- pin structure capable of hybridizing with the tertiary nucleic acids with the formation of GTN (see. US-A 5,798,210, Fig. 8). This alternative would in any case only be considered if in step (e) in which the protecting group is removed, conditions are chosen that do not denature, does not mean the melting of the double strands consisting kleotiden from optionally extended Oligonu- and tertiary nucleic acids perform. In step (e) carried out under denaturing conditions (eg by use of stronger bases), one will preferably make use of the other measures.

As part of the measures described, the length of the oligonucleotides plays a subordinate role. In general, the oligonucleotides have a length of less than 100 or less than 50 nucleotide building blocks, so that also use generally of nucleic acids: can speak (here polymeric nucleotides comprising more than three nucleotide). Single-stranded oligonucleotides of length greater than 45 nucleotide building blocks are difficult to handle due to nonspecific interactions if they have no sequence allows Haiφins. By the ability to form Haiφins, non-specific interactions can be reduced by competition. Be double-stranded oligonucleotides are used, the length of the oligonucleotides hardly therefore plays a role (see also Fig. 3).

All measures described have the consequence that the tertiary nucleic acids have a double-stranded portion which skriptase enables strand extension in the complementary strands of the tertiary nucleic acids (GTN) by a DNA polymerase or reverse transit.

In the nucleotide. in step (c) is incorporated is complementary to the opposite strand, it is a entschützbares Abbruchnukleotid. Suitable Abbruchnukleotide are known for example from US-A 5,798,210. Canard and Sarfati (Gene 148 (1994) 1-6) describe 3 '-veresterte nucleotides that contain an abstractable together with the protecting group fluorophore. This nucleotide may be inkoφoriert, albeit with low efficiency of different polymerases in a growing strand and then act as Abbruchnukleotide, so let no further strand extension to. The esters described may be split off enzymatically or alkaline, so that free, more Nukleotidinkoφoration arise permitting 3 '-OH groups. However, the Esterspaltung is very slow (within 2 hours), so that the compounds described for sequencing longer DNA segments (eg more than 20 bases) are unsuitable. As long as the protecting group at the 3'-OH or optionally 2'-OH position (see below) is attached, the quaternary nucleic acid extended by this nucleotide no longer a substrate for a nucleic acid polymerase. It is only the removal of the protecting group in step (e) makes a further extension of the quaternary nucleic possible. The Working Party also usually wears a molecular roup that allows identi- fication of the incorporated nucleotide and so the sequencing of the growing nucleic acid strand and leaves the nucleotide with the removal of the protective group. However, the identifying group of molecules can also be at another location of the nucleotide may be bound to the Example of the base. In this case, it is necessary to erase after step (d) in step (e) is the signal of the identifying group of molecules. This can be done generally in two ways. For example, in the case of a fluorophore molecule the group can be changed by fading. In addition, the identifying group of molecules may also be removed, for example by photochemical cleavage of a photolabile bond. Is the identifying group of molecules not connected with the protective group and the identifying molecule group is cleaved for signal cancellation, so the binding of protective group on the nucleotide and the binding of the identifying molecular group to the nucleotide should preferably be chosen so that both groups are split off in a reaction step can. Preferably, each bears the candidate for the installation of four nucleotide (G, A, T, C) other identifying molecular group. In this case, the four kinds of nucleotides in step (c) can be offered simultaneously. Wear different or so-even all nucleotides same identifying molecular group, step (c) to dismantle four stages in a rule in which the nucleotides of one type (G, A, T, C) are sold separately.

In the molecule roup is, for example, a fluorophore or a chromophore. The latter could be Absoφtionsmaximum in the visible or infrared frequency range. In step (d) taking place detection takes place both spatially and temporally resolved, so that the islands located on the surface of quaternary nucleic acids can be sequenced in parallel.

As the protecting group of the nucleotide in step (c) is meant a chemical substituent which prevents the further strand extension after incorporation of the nucleotide at the 3 'position. The protecting group may 'occupy position, thus be connected to the C-3 of the ribose or the protected 3' to the protective shield 3 position, thus hindering the Srangverlängerung sterically. In the latter case the protecting group would be joined at the adjacent position, particularly at the C-2 of the ribose with the nucleotide.

In a further embodiment of the inventive method in step (al) primer or nucleic acid molecules are used with flanking sequence portions having self-complementary regions.

In a further embodiment of the method in step (b), the tertiary nucleic acids cut by a restriction endonuclease prior to be ligated to the ends generated in this way oligonucleotides which are capable of forming a Haiφinstruktur. It is to the explanations of step (b) Action 2 referred to page 9, and in particular the explanation of the term oligonucleotide.

In a further embodiment of the inventive method capable of forming a Haiφinstruktur oligonucleotides are single stranded. Single stranded here means not consistently double-stranded. The oligonucleotides are not so before as a heterodimer. This is the case for example in FIG. 2.

In a further embodiment of the inventive method capable of forming a Haiφinstruktur oligonucleotides are double-stranded. The oligonucleotides are therefore present as a heterodimer. This is for example the case 3 in Fig.. In a further embodiment of the inventive method, in step (b) single-stranded oligonucleotides which are capable of forming a hybridized to Haiφinstruktur tertiary nucleic acids, nucleic acids and before tertiary aforesaid mono- zelstängige oligonucleotides are ligated. This is the case for example in FIG. 2.

In this case, should however be noted that the hybrid formation is often unstable (for example, if existing 4 nucleotide overhangs are hybridized) so that hybrid formation and ligation follow one another directly. In a further embodiment of the method according to the invention, be linked to tertiary nucleic acids by ligation in step (b) single-stranded oligonucleotides which are capable of forming a Haiφinstruktur. Here, the ligation non-sticky end (blunt ends) of the question. This does not require any previous hybrid formation.

In a further embodiment of the inventive process in step (a, al), the primer molecules irreversibly immobilized by forming a covalent bond to a surface.

In a further embodiment of the method according to the invention, the base bears in step (c) the molecular group that enables the identification of the nucleotide.

In a further embodiment of the inventive method, the currency protecting group transmits in step (c) the nucleotide at the 3'-OH position.

In a further embodiment of the inventive method, the protecting group to a cleavable ester, ether, anhydride or peroxide group.

In a further embodiment of the inventive method, the protecting group is connected via an oxygen-metal bond with the nucleotide.

In a further embodiment of the inventive method (s) in step is the removal of the protecting group by a complex-forming ion, preferably by cyanide, thiocyanate, fluoride, or ethylene diamine tetraacetate.

In a further embodiment of the inventive method, the protecting group in step (c) a fluorophore, and in step (d) the nucleotide is symmetrical identified fiuorome-. In a further embodiment of the inventive method (s) is deprotected in step photochemically.

The invention is further described by the drawing, the drawing sheets are provided with consecutive numbers (1 / 10-10 / 12). It shows

Figure 1 shows the amplification of individual nucleic acid molecules using surface bound primers to islands from each identical amplified nucleic acid molecules comprising a drawing sheet (1/12). Figure 2 shows the sequencing of surface-bound amplification, comprising Figures 2a, 2b, 2c .. Figure, the drawing pages 12.2 to 12.4...; Fig. 3 taken from the provision of a GTN by forming a Haiφinstruktur in sequence portions linkers comprising Figures 3a, 3b, 3c to the drawing pages 12.5 to 12.7...; Figure 4 illustrates the parallel sequencing on a surface, comprising a drawing sheet (8/12). Figure 5 shows the assembly of the detection and identification results to contiguous sequences comprising a character sheet (9/12). Fig. 6 is the provision of primary nucleic acids for use in the expression analysis, comprising a drawing sheet (10/12);

Figure 7 shows the provision of primary nucleic acids for sequencing of genomic clones comprising a drawing sheet (11/12). Fig. 8 shows the result of Amplikation individual nucleic acid molecules according to Fig. 1, comprising a drawing sheet (12/12).

Fig. 1 shows the amplification of individual nucleic acid molecules using surface bound primers to islands from each identical amplified nucleic acid molecules, wherein the irreversible immobilization of forming a Primeφaar in detail the Oligonukleoti- 1,

2, the hybridization of the primary nucleic acids with the surface bound primers,

3, the formation of secondary nucleic acids by continuous extension of the primers, 4, the removal of the non irreversibly bound primary nucleic acid molecules and the amplification of the secondary nucleic acids,

5 denotes islands each having identical tertiäreren nucleic acid molecules. Fig. 2 illustrates the sequencing of surface-bound amplification products, wherein

1 the release of the amplification products by restriction digestion of the amplification products (underlined: the recognition site for the Restriktionsendonu- klease Sphl) (SEQ ID NO: 4);

2 dephosphorylation;

3, the ligation of an oligonucleotide Haiφin (bold)

4, the removal of the irreversibly immobilized nucleic acid strand (SEQ ID NO: 6); 5 installation and identification of a first protected nucleotide;

6, the removal of protecting group and mark group with recovery of a free 3 '-OH group;

7 installation and identification of a second protected nucleotide;

8 shows the repetition of steps 5 and 6. FIG.

Fig. 3 shows the provision of a GTN by forming a Haiφinstruktur in sequence segments originate from the linker. The nucleic acid to be sequenced (restriction fragment with two different ends, one thereof by the restriction endonuclease N / αlll generated) is shown hatched. CATG, generated by restriction endonuclease Nlalϊl overhang; GCATGC, recognition site for restriction endonuclease Sph I (contains the recognition site for N / αlll, CATG); ΝΝΝΝΝΝΝΝΝΝ and MMMMMMMMMM, "inverted repeats" (mutually complementary sequences, the intramolecular refolding of a single strand allow); XXXXX and YYYYY, spacer region to the surface Specifically 1 describes the ligation of a linker with "inverted repeat" and S bl interface. fragment to be sequenced;

2, the denaturation and hybridization with immobilized to a surface primer;

3, the amplification with two primers immobilized on the surface (counter primer not shown);

4, the "half-page loosening" of the amplification products from the surface by restriction endonuclease SphI (arrows);

5, the denaturation and removal of non-immobilized on the surface of the strand; 6, the renaturation, forming a Haiφins, the beginning of the sequencing by Inkoφoration entschützbarer Abbruchnukleotide. Fig. 3 shows a preferred Norge Hens manner to provide serving as Sequenzieφrimer opposite strands of the tertiary nucleic acids, GTN, by the treatment with a first restriction endonuclease equipped (in Fig. 3 about the recognition sequence CATG comprising) with overhanging ends of nucleic acid molecules, initially in a ligation with flanking sequence sections are provided in the form of double-stranded linker molecules, which include first and second self-complementary regions have distally adjacent to this interface or a recognition sequence for a second restriction endonuclease. Preferably, these are as in Fig. 3 by an interface whose inner bases identical to the bases of said overhang on the same line (in Fig. 3, the base sequence CATG), at least one of the outer bases, however, as appropriate for the , said overhang sequence before or after ligation of flanking base differs. For example, shown in Fig. 3, that the related for ligation overhang "CATG" is flanked by ligation at its 3 'end from the base "T". If now after the amplification of the nucleic acid molecules in step (a5) using a Primeφaars from which a primer can hybridize with one strand of said linker molecules, cut by a second restriction endonuclease which has, for example, the recognition sequence "GCAGTC" and this recognition sequence was flanking in said sequence sections (that is, as part of the sequence of the attached linker) provided, a cut is made in the provided sequence portions. After the removal of the then no longer irreversibly bound to the surface strand of the 3 'terminus of the immobilized remaining strand can intramolecularly refold to a Haiφin. Here, is preferred that, as shown in Fig. 3, limits the recognition sequence of said first and second restriction endonuclease inserted directly to the self-complementary regions, so that these areas by said ligation by said two detection sstellen common bases are extended. Here, the extended self-complementary regions there have a mismatch, where after the ligation, the overhang sequence flanking base (or bases) is different from the recognition sequence of the second restriction endonuclease or different (in Fig. 3 is a G / T mismatch) , At the same time ensures the Norge Hens described here, in which the recognition site of the first restriction endonuclease is a part of the longer recognition site of the second restriction endonuclease that the tertiary nucleic acid molecules may have no internal recognition sites for the second restriction endonuclease during the incubation with the second restriction endonuclease, but only exactly once be cut in the flanking sequences.

FIG. 4 describes the parallel sequencing on a surface. "Islands" of identical nucleic acid molecules are in this Figure simplified by a single strand symbolizes. Specifically, Figure 1 shows the attachment of a Sequenzieφrimers, installation of the first Abbruchnukleo- TIDS and parallel detection and identification of the respective first nucleotide building block,

2, the removal of protecting group, and label group of the first nucleotide TIDS, installation of the second Abbruchnukleotids and parallel detection and

Identification of the respective second nucleotide building;

3, the detection and identification result of the first base;

4, the detection and identification result of the second base.

FIG. 5 describes the assembly of the detection and identification results to contiguous sequences,

1, the detection and identification results of the first base,

2, the detection and identification results of the second base,

3, the detection and identification results of the n-th base 4 referred to the assembled sequences of the nucleic acid molecules in each of the islands.

Fig. 6 shows the provision of primary nucleic acids for use in the expression analysis, in which the individual

1 cDNA synthesis with biotinylated primer binding the double stranded cDNA to streptavidin-coated surface;

2 shows the restriction with the first enzyme (RE1), washing away the released fragments, and the second restriction enzyme with the second (RE2);

3, the ligation of two different linker; 4 mRNA;

5 double immobilized on solid phase cDNA;

which is flanked by two different "overhanging" ends 6, a cDNA fragment,

7 one of two different linkers (Ll, L2) is flanked cDNA fragment.

Fig. 7 shows the provision of primary nucleic acids for sequencing genomic clones, wherein in each

1 is a restriction of a genomic clone in parallel, each with two different restriction endonucleases (RE1-2 or rE3-4), ligation of various linker (L 1 -4);

2 a genomic clone; 3 denotes two overlapping sets with linkers ligated fragments. Symmetrically flanked by identical linkers fragments (crossed) can not be sequenced.

Fig. 8 shows the result of amplification of individual nucleic acid molecules by means of surface bound primers to islands from each identical amplified nucleic acid molecules, visualized by staining with SYBR Green I.

The invention will be explained in the following by examples.

Example 1:

Preparation of nucleic acid molecules

4 ug of total RNA from rat liver was precipitated with ethanol and dissolved in 15.5 .mu.l of water.

There was added 0.5 ul of 10 uM cDNA primer CP28V (5'ACCTACGTGCAGATTTTTTTTTTTTTTTTTTV-3 ', SEQ ID NO: 1) was added, denatured for 5 minutes at 65 ° C and placed on ice. The mixture was 5χ with 3 .mu.l of 100 mM dithiothreitol (Life Technologies GmbH, Karlsruhe, Germany), 6 ul Superscript buffer (Life Technologies GmbH, Karlsruhe, Germany), 1.5 ul of 10 mM dNTPs, 0.6 40 .mu.l RNase inhibitor (U / ul ; Roche Molecular Biochemicals) and 1 .mu.l of Superscript II (sets 200 u / ul, Life Technologies) and comparable to cDNA first-strand synthesis for 1 hour at 42 ° C. For second strand synthesis 48 ul second strand buffer (see FIG. Ausubel et al., Current Protocols in Molecular Biology (1999), John Wiley & Sons), 3.6 ul of 10 mM dNTPs, 148.8 ul H 2 O, 1.2 .mu.l RNase H (1.5 U / ul, Promega) and 6 .mu.l DNA polymerase I (New England Biolabs GmbH Schwalbach, 10 U / ul) was added and the reactions for 2 hours at 22 ° C. It was extracted with 100 ul phenol, then with 100 ul chloroform and precipitated with 0.1 vol. Nafriumacetat pH 5.2 and 2.5 vol. Ethanol. After centrifugation for 20 minutes at 15,000 g and washed with 70% ethanol, the pellet was resuspended in a restriction mixture of 15 ul lOx Universal buffer, 1 ul dissolved Mbol and 84 ul HO and the reaction incubated for 1 hour at 37 ° C. It was then extracted with phenol and chloroform and precipitated with ethanol. The pellet was resuspended in a ligation mixture consisting of 0.6 ul lOx ligation buffer (Roche Molecular Biochemicals), 1 ul 10 mM ATP (Roche Molecular Biochemicals), 1 ul linker ML2025 (manufactured by hybridization of oligonucleotides ML20 (5'TCACATGCTAAGTCTCGCGA-3 ', SEQ ID NO: 5) and LM25 (5'GATCTCGCGAGACTTAGCATGTGAC-3 ', SEQ ID NO: 7), ARK) dissolved 6.9 ul H 2 O and 0.5 ul T4 DNA ligase (Roche Molecular Biochemicals) and the ligation overnight at 16 ° C. The ligation reaction was made up with water to 50 .mu.l, with phenol, then extracted with chloroform and, after addition of 1 .mu.l glycogen (20 mg / ml, Roche Molecular Biochemicals) with 50 ul 28% polyethylene glycol 8000 (Promega) /! 0 mM MgCl2 like. The pellet was washed with 70% "ethanol and taken up in 100 .mu.l of water.

Example 2: Coating with oligonucleotides

Lyophilized, at its 5 '-end Aminolink-group-carrying oligonucleotides amino M13rev (5'-amino-CAGGAAACAGCGATGAC-3 \ SEQ ID NO: 8) and amino-T7 (5'-TAATACGACTCACTATAGG-amino-3', SEQ ID NO : 10) (ARK Scientific GmbH, Darmstadt) were added to a final concentration of 1 mM in 100 mM Natriumcarbonatpuf- fer pH. 9 Microscopy glass slides ( "slides"; neoLab Migge Laboratory-Vertriebs GmbH, Heidelberg) were cleaned for 1 hour in chromic acid and then washed 4x with distilled water After air drying, the slides were 5 minutes dissolved in a l% solution of 3-aminopropyltrimethoxysilane. ( "Fluka": Sigma Aldrich Chemie GmbH, Seelze) 5% water treated in 95% acetone /. The mixture was then washed ten times, each for 5 minutes in acetone and heated to 110 ° C for 1 hour. methylformamide in a solution of 10% pyridine in dry di- (Merck KGaA, Darmstadt) After inserted: Then the slides for 2 hours in 0.2% 1,4-phenylene (Sigma Aldrich Chemie GmbH, Seelze "Fluka") were. 5 washes in methanol and 3 washes in acetone, the slides were air-dried and directly further processed for loading stratification. Adhesive "frame SeaP'-frames for 65 μl- reaction chambers (MJ Research Inc., Watertown, Minnesota, USA) were applied 65 ul oligonucleotide solution were pipetted into the thus formed reaction chambers, and the chambers were adhering a polyester cover sheet (MJ Research Inc.) sealed in the absence of air bubbles. the exact position of the reaction chamber was marked with a permanent felt tip pen on the bottom of the slides. the binding of the oligonucleotides via Aminolink to the surface of the activated slides was 4 hours instead of at 37 ° C. Subsequently, the Adhesive frame removed and rinsed off the slides with deionized water. To inactivate any remaining reactive groups, the slides for 15 minutes were placed in temperature-controlled at 50 ° C blocking solution (50 mM ethanolamine ( "Fluka": Sigma Aldrich Chemie GmbH, Seelze), 0.1 M Tris pH 9 ( "Fluka": Sigma Aldrich Chemie GmbH, Seelze), 0.1% SDS ( "Fluka". treated Sigma Aldrich Chemie GmbH, Seelze) to remove non-covalently bound oligonucleotides, the slides for 5 minutes in 800 ml 0.1 x SSC / 0.1 were % SDS (see FIG. Ausubel et al., Current Protocols (in Molecular Biology 1999), John Wiley & Sons) cooked. The slides were washed with deionized water and air dried.

Example 3:

Coating with oligonucleotides Lyophilised, at its 5 '-end bearing Aminolink groups oligonucleotides amino M13rev (5'-amino-CAGGAAACAGCGATGAC-3' sequence of nucleotides as shown in SEQ ID NO: 8) and amino-T7 (5'-amino- TAATACGACTCACTATAGG 3 ', sequence of nucleotides as shown in SEQ ID NO: 10) (ARK Scientific GmbH, Darmstadt) were added to deionized water to a final concentration of 100 pmol / ul. Depending 1.4 .mu.l of this primer solutions were mixed with 32.2 .mu.l of water and 35 ul 2x binding buffer (300 mM sodium phosphate pH 8.5). Self-adhesive "Frame SeaF'-frames for 65 μl- reaction chambers (MJ Research Inc., Watertown, Minnesota, USA) were applied to" SD link activated slides "(activated for binding amino-modified nucleic acids of glass object carrier (Surmodics, Eden Prairie, Minnesota, USA) was applied. 65 ul Oligonu- kleotid solution were pipetted into the thus formed reaction chambers, and the chambers were (MJ Research Inc. by sticking a polyester cover sheet, Watertown, Minnesota, USA) with the exclusion of bubbles sealed. the exact position of the reaction chamber was marked with a permanent felt tip pen on the bottom of the slides. the binding of the oligonucleotides via Aminolink to the surface of the activated slides was held at room temperature overnight. Subsequently, the adhesive frame were removed and the slides with deionized water rinsed off. in order to inactivate any remaining reactive groups, the slides for 15 minutes were at 50 ° C tem perierter blocking solution (50 mM ethanolamine ( "Fluka": Sigma Aldrich Chemie GmbH, Seelze), 0.1 M Tris pH 9 ( "Fluka": Sigma Aldrich Chemie GmbH, Seelze), 0.1% SDS ( "Fluka": Sigma Aldrich Chemie GmbH, Seelze) treated. In order to remove non-covalently bound oligonucleotides, the slides for 5 minutes in 800 ml 0.1 x SSC / 0.1% SDS (see Ausubel et al., Current Protocols in Molecular Biology (1999), John Wiley & Sons.) Were boiled. The slides were washed with deionized water and air dried.

Example 4:

Plasmids pRNODCAB (containing bases 982-1491 of the transcript of Ornithindecar- decarboxylase from rat AC number J04791 cloned into vector pCR II (Invitrogen BV, Groningen, Netherlands) and pRNHPRT (contains bases 238-720 of the transcript hybrid poxanthinphosphoribosyltransferase rat , AC-number M63983, cloned into vector pCR II (Invitrogen)) were linearized by each 1 ug plasmid lx in a volume of 20 ul restriction buffer H ( "Roche Molecular Biochemicals": Roche Diagnostics GmbH, Mannheim, Germany) with 5 U of restriction enzymes BglII and Scαl (Roche Molecular Biochemicals) was incubated for 1.5 hours at 37 ° C. then, an amplification of the vector-insert was performed by adding 1 ul of each restriction mixtures in a volume of 100 ul PCR buffer II (Perkin- Elmer, Foster City, California, USA) with 4 .mu.l 10 mM primer T7 (5'-TAATACGACTCACTATAGG-3 \ SEQ ID NO: 10), 4 ul 10 mM primer Ml 3 (5'-CAGGAAACAGCGATGAC-3 ', SEQ ID NO : 8) (ARK), 4 ul 50 mM MgCl 2 ( "Fluka": Sigma Aldrich Chemie GmbH, Seelze), 5 .mu.l of dimethyl sulfoxide ( "Fluka": Sigma Aldrich Chemie GmbH, Seelze), 1 ul 10 mM dNTPs (Roche Molecular Biochemicals), and 1 ul of AmpliTaq DNA polymerase (5u / ul ; Perkin-Elmer) was added. Subsequently, the reactions were (Perkin-Elmer) subjected in a Gene Amp 9700 thermal cycler a Temperatuφrogramm consisting of 20 cycles of denaturation for 20 seconds at 95 ° C, primer annealing for 20 seconds at 55 ° C and primer extension for 2 minutes at 72 ° C. The amplification products were analyzed by electrophoresis on a 1.5% Aga rosegel to their right size out. To remove uninkoφorierter primer reactions using QiaQuick columns (Qiagen AG, Hilden, Germany) were prepared according to the manufacturer ANGA ben cleaned and eluted in 50 ul of deionized water.

Example 5:

amplification

For the attachment of the prepared in Example 2 nucleic acids to glass slides were arrival nealing mixtures undiluted from each 1 ul or in parallel batches, 1:10, 1: 100 or 1: 1000 diluted with water amplification solutions, additional 4 ul 50 mM MgCl 2 - solution, each 1 ul bovine serum albumin (20 mg / ml; Roche Molecular Biochemicals) mM dNTPs and 1 ul AmpliTaq lx in a total volume of 100 .mu.l of PCR buffer II prepared per 5 .mu.l of dimethyl sulfoxide, each 1 ul 10th Observing the Filzschreiber- marks on the Slide Base Frame-Seal chambers were applied in the conditions used for Oligonu- kleotid coating positions on the prepared in Example 1 slides. Then each 65 ul of the annealing mixture were pipetted into the reaction chambers and the chambers sealed as above. The slides were placed on the heating block of a UNO Il-in situ thermal cycler (Biometra biomedical Analytik GmbH, Göttingen, Germany), covered with a pad of paper towels and pressed against the heating block by means of the height-adjustable heated lid. For annealing and subsequent primer extension following Temperatuφrogramm was applied: denaturation 30 seconds at 94 ° C, annealing for 10 minutes at 55 ° C, primer extension for 1 minute at 72 ° C. After the reaction, the reaction chambers were removed and the slides were rinsed with de-ionized water. To remove the non-covalently bound strands boiled for 1 minute in 800 ml 0.1 x SSC / 0.1% SDS, the slides rinsed with water and air dried. To make a compartmentalized amplification of the support-bound nucleic acid molecules, reaction chambers were again to the previously selected positions applied and applied 65 ul of a amplification mixture to-sammengesetzt as follows: 4 ul 50 mM MgCl 2, 1 ul bovine serum albumin (20 mg / ml) , 5 .mu.l of dimethyl sulfoxide, 1 ul AmpliTaq (5 U / ul), 1 ul 10 mM dNTPs, lx in 100 ul PCR buffer II After sealing of the chambers has been applied to the following in situ thermocycler Temperatuφrogramm. denaturation for 20 seconds at 93 ° C, primer annealing 20 seconds at 55 ° C, extension for 1 minute at 72 ° C, for 50 cycles. After the end of amplification, the chambers were removed and rinsed the slides with water and air dried. pipetted onto the slides and covered with cover slips # 2 (MJ): (10,000 in water solution ser 1 Molecular Probes) for detecting the resulting compartmentalized by clonal amplification islands 40 ul SYBR Green I solution. Detection was performed on a confocal microscope TCS-NT (Leica Microsystems Heidelberg GmbH, Heidelberg) at an excitation wavelength of 488 nm and a detection wavelength of 530 nm. It could clonal islands compartmentalized amplified nucleic acid molecules are detected, in a random array on the slide surface loading area of ​​the reaction chambers distributed (see Fig. Fig. 8). In the range of reaction chambers in which had been bound as a negative control either no oligonucleotides on the support or in which the amplification was carried out without prior hybridization of template molecules, while not originating from clonal islands signals were detected. Continued to show the comparison of the slide surfaces in the area of ​​reaction chambers in which different concentrations of template was used, a clear dependence on the number of clonal islands formed on the amount of inserted molecules.

Example 6: In order to identify the nucleic acid molecules in the detected clonal islands, the slides were after the detection of the stained using SYBR Green double-stranded DNA

10 minutes discolored water. Then reaction chambers were once again at the same

Positions as previously glued and a restriction mixture consisting of 12 ul lOx

■ Universal buffer (Stratagene GmbH, Heidelberg), 1 ul bovine serum albumin, 3 .mu.l restrictive endonuclease Mbo I (1 U / ul; Stratagene) and 64 .mu.l of water, was added by pipette. For restriction of the nucleic acid molecules by means of the internal Λ BOI interface was 1.5 h at 37 ° C, then the reaction chambers were removed and the slides washed with water. The non-covalently bound to the carrier strand glass fragments were duch denaturation for 2 minutes in 800 ml boiling 0, lχ SSC / 0.1% SDS removed. After washing in water and air drying new reaction chambers were applied. Was per hybridization experiment a hybridization solution of 8 ul lOx PCR buffer II, 3.2 .mu.l 50 mM MgCl2, 2 ul 100 pmol / ul oligonucleotide probe Cy5-HPRT (5'-Cy5-TCTACAGTCATAGGAATGGACCTATCACTA-3 \ SEQ ID NO: 3; ARK ), 2 ul 100 pmol / ul oligonucleotide Cy3-ODC (5'-Cy3 ACATGTTGGTCCCCAGATGCTGGATGAGTA-3 ', SEQ ID NO: 2) and 65 .mu.l of water. 65 ul thereof was added in the respective reaction chamber for each hybridization experiment and hybridized at 50 ° C for 3 hours. After completion of the hybridization, the reaction chambers were removed and the slides for 5 minutes at room temperature in 30 ml of 0, SSC / 0.1% SDS lχ washed. The slides were briefly rinsed with distilled water, air dried and used for detection. Detection was performed as above using a confocal laser microscope. As excitation wavelengths 568 nm and 647 nm were used were detected signals at 600 nm and at 665 nm. It has been shown that the previously detected with SYBR Green clonal islands in part by the probe Cy3-ODC and in part by the probe Cy5 -HPRT were detected.

Example 7: Expression analysis by massively parallel sequencing of nucleic acid molecules

The ligation products obtained in Example 1 were diluted 1: 1000 with water and 1 ul of this dilution as described in Example 5 for 50 cycles compartmentalized amplitude-fied. For this purpose were as described with the amplification primers amino-CP28V (5'-amino-ACCTACGTGCAGATTTTTTTTTTTTTTTV-3 'sequence of nucleotides as shown in SEQ ID NO: 1) and amino ML20 (5'-amino-3-TCACATGCTAAGTCTCGCGA \ sequence of nucleotides as shown in SEQ ID NO: 5) coated glass slides used. For one-sided separation of the amplification products from the support, the Amplifikationsmi- research was replaced by a restriction mixture consisting of 12 ul lOx Universal buffer (Stratagene), 1 ul bovine serum albumin, 4 .mu.l restriction endonuclease Mbo I, in a final volume of 65 ul. After incubation at 37 ° C for 2 h, the restriction mixture was replaced by a dephosphorylation of 1 U alkaline phosphatase, Arctic shrimp (Amersham) in 65 ul of the reaction buffer supplied. After incubation for 1 hour at 37 ° C and inactivation for 15 minutes at 65 ° C and the reaction chambers dephosphorylation were removed, the slides washed thoroughly with distilled water, applied again and reaction chambers filled with 65 ul of a gationsmischung Li, consisting of 3 U T4 DNA ligase (Roche Diagnostics) and 500 ng of the S'-phosphorylated end-Haiφin Sequenzieφrimer SLP33 (5'TCTTCGAATGCACTGAGCGCATTCGAAGAGATC-3 ', SEQ ID NO: 9) in 65 ul of ligation buffer supplied. It was ligated for 14 hours at 16 ° C, then were league tion mixture and reaction chambers removed. To remove the non-covalently bound to the carrier strand glass fragments l was for 2 minutes in 800 ml boiling 0, treated SSC / 0.1% SDS and washed with distilled water. To prepare suitable entschützbarer Abbruchnukleotide dATP, dCTP, dGTP and dTTP (Roche Molecular Biochemicals) were esterified at their 3'-OH group of 4-aminobutyric acid. These derivatives were incubated with the fluorescent FAM groups (dATP, dCTP) and ROX (dGTP, dTTP) marked (Molecular Probes Inc., Eugene, Oregon, USA). For determining in parallel the first base reaction chambers were re-applied to the slides and a primer extension mixture of 1 mM FAM-dATP, 1 mM ROX-dGTP and 2 U Sequenase (United States Biochemical Coφ., Cleveland, Ohio, USA) in 65 ul reaction onspuffer (40 mM Tris-HCl pH 7.5, 20 mM MgCl 2, and 25 mM NaCl) were charged. After

Incubation at 37 ° C for 5 minutes was washed with reaction buffer and the

Laser Scanning Microscope detected. Excitation wavelengths were 488 nm and 568 nm, detected at 530 nm and at 600 nm. After the detection Primerex- tensionsmischung was added again, now containing the remaining two labeled nucleotides, FAM-ROX and dCTP-dTTP. After Inkoφoration was again washed and detected, and the protecting groups were removed by enzymatic cleavage. To this was treated with 5 mg / ml Chirazyme L lipase (Roche Diagnostics) in 100 mM potassium phosphate buffer pH 9 for 1 h at 35 ° C. Then the sequencing was performed as described above for 15 additional cycles performed.

Claims

claims
1. A method for parallel sequencing of at least two different nucleic acids contained within a nucleic acid mixture, wherein
(A) is a surface provided, comprising islands of nucleic acids each variety, nucleic acids tertiary;
(B) are opposite strands of the tertiary nucleic acids, GTN provided;
(C) the GTN be extended by one nucleotide, wherein
- the nucleotide carrying a protective group at the 2'-OH position or at the 3'-OH position, which prevents further extension
- the nucleotide carries a molecular group which enables the identification of the nucleotide;
(D) identifying the incorporated nucleotide;
(E) the protective group is removed, and removes the molecule group used for the identification of the incorporated nucleotide or modified, and
(F) Step (c) and subsequent steps be repeated until the desired sequence information has been obtained.
2. The method of claim 1, wherein in step (a)
(Al) is a surface provided, to which at least primer molecules of a first primer and a second primer, and optionally a nucleic acid mixture, are irreversibly immobilized comprising the nucleic acid molecules with which both primers are capable of hybridizing, wherein both primers form a Primeφaar;
(A2) the nucleic acid molecules of the nucleic acid mixture are hybridized with one or two primers of the same Primeφaares;
(A3) the irreversibly immobilized primer molecules are extended complementary to the opposite strand to form secondary nucleic acids;
(A4) is provided in the surface of a Nukleinäuremolekülen that are not bound by irreversible immobilization to the surface, freed form;
(A5), the secondary nucleic acids are amplified to form tertiary nucleic acids;
3. The method of claim 2, wherein in step (al) a surface to which at least one Primeφaar forming primer molecules have been irreversibly immobilized, is provided.
4. The method according to any one of claims 1 to 3, characterized in that in step (al) primer or nucleic acid molecules are used with flanking sequence portions having self-complementary regions.
5. The method according to any one of claims 1 to 3, characterized in that in step (b) the tertiary nucleic acids are cut by a restriction endonuclease prior to be ligated to the ends generated in this way oligonucleotides which are capable of forming a Haiφinstruktur.
6. A method according to claim 5, characterized in that the capable of forming a Haiφinstruktur oligonucleotides are single stranded.
7. A method according to claim 5, characterized in that the capable of forming a Haiφinstruktur oligonucleotides are double-stranded.
8. A method according to any one of claims 1 to 3, characterized in that in step (b) single-stranded oligonucleotides which are capable of forming a Haiφinstruktur, are hybridized to nucleic acids tertiary before tertiary nucleic acids and the aforementioned single-stranded oligonucleotides are ligated.
9. A method according to any one of claims 1 to 3, characterized in that in step
(B) single-stranded oligonucleotides which are capable of forming a Haiφinstruktur, be linked to tertiary nucleic acids by ligation.
10. The method according to any one of claims 2 to 9 characterized in that in step (al) the primer molecules are irreversibly immobilized by forming a covalent bond to a surface.
11. A method according to any one of claims 1 to 10, characterized in that in step
(C) the base carries the molecule group, which enables the identification of the nucleotide.
12. The method according to any one of claims 1 to 11, characterized in that in step (c), the protecting group transmits the molecule group, which enables the identification of the nucleotide.
13. The method according to any one of claims 1 to 12, characterized in that in step (c), the protecting group carries the nucleotide at the 3'-OH position.
14. A method according to any one of claims 1 to 12, characterized in that in step (c), the protecting group carries the nucleotide at the 2'-OH position.
15. The method according to any one of claims 1 to 14, characterized in that in step (c), the protecting group has a cleavable ester, ether, anhydride or peroxide group.
16. The method according to any one of claims 1 to 14, characterized in that in step (c), the protecting group is connected via an oxygen-metal bond with the nucleotide.
17. The method according to claim 16, characterized in that in step (e) the removal of the protecting group by a complex-forming ion, preferably by cyanide, thiocyanate, fluoride, or ethylene diamine tetraacetate.
18. The method any one of claims 1 to 14, characterized in that in step (e), the protective group is cleaved photochemically.
19. A method according to any one of claims 1 to 18, characterized in that in step (c), the protecting group comprises a fluorophore and, in step (d) the nucleotide is fluorescently rometrisch identified.
EP20000990813 1999-12-23 2000-12-22 Method for carrying out the parallel sequencing of a nucleic acid mixture on a surface Withdrawn EP1244782A2 (en)

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DE1999162893 DE19962893A1 (en) 1999-12-23 1999-12-23 Parallel sequencing of nucleic acids in a mixture, useful e.g. for detecting mutations, comprises a series of single-base extension reactions using protected, labeled nucleotides
DE10051564 2000-10-18
DE2000151564 DE10051564A1 (en) 2000-10-18 2000-10-18 Parallel sequencing of nucleic acids in a mixture, useful e.g. for detecting mutations, comprises a series of single-base extension reactions using protected, labeled nucleotides
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Families Citing this family (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE60127162D1 (en) 2000-10-06 2007-04-19 Univ Columbia Massive Parallel decoding method of DNA and RNA
US9708358B2 (en) 2000-10-06 2017-07-18 The Trustees Of Columbia University In The City Of New York Massive parallel method for decoding DNA and RNA
DE10106320A1 (en) * 2001-02-09 2002-08-22 Axaron Bioscience Ag Production and use of random arrangements of clonal nucleic acid islands on a surface
US7414116B2 (en) 2002-08-23 2008-08-19 Illumina Cambridge Limited Labelled nucleotides
US7057026B2 (en) * 2001-12-04 2006-06-06 Solexa Limited Labelled nucleotides
GB0129012D0 (en) 2001-12-04 2002-01-23 Solexa Ltd Labelled nucleotides
WO2004018497A3 (en) 2002-08-23 2004-06-17 Colin Barnes Modified nucleotides for polynucleotide sequencing
DE10224339A1 (en) * 2002-05-29 2003-12-11 Axaron Bioscience Ag A method for highly parallel sequencing of nucleic acids
US7169560B2 (en) 2003-11-12 2007-01-30 Helicos Biosciences Corporation Short cycle methods for sequencing polynucleotides
EP2620510B1 (en) 2005-06-15 2016-10-12 Complete Genomics Inc. Single molecule arrays for genetic and chemical analysis
GB0514935D0 (en) 2005-07-20 2005-08-24 Solexa Ltd Methods for sequencing a polynucleotide template
GB0517097D0 (en) 2005-08-19 2005-09-28 Solexa Ltd Modified nucleosides and nucleotides and uses thereof
US7666593B2 (en) 2005-08-26 2010-02-23 Helicos Biosciences Corporation Single molecule sequencing of captured nucleic acids
US8399188B2 (en) 2006-09-28 2013-03-19 Illumina, Inc. Compositions and methods for nucleotide sequencing
US7883869B2 (en) 2006-12-01 2011-02-08 The Trustees Of Columbia University In The City Of New York Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators
WO2009046094A1 (en) 2007-10-01 2009-04-09 Nabsys, Inc. Biopolymer sequencing by hybridization of probes to form ternary complexes and variable range alignment
EP2209911B1 (en) 2007-10-19 2013-10-16 The Trustees of Columbia University in the City of New York Dna sequencing with non-fluorescent nucleotide reversible terminators and cleavable label modified nucleotide terminators and a deoxyinosine analogue with a reversible terminator group
US20110014611A1 (en) * 2007-10-19 2011-01-20 Jingyue Ju Design and synthesis of cleavable fluorescent nucleotides as reversible terminators for dna sequences by synthesis
US8262879B2 (en) 2008-09-03 2012-09-11 Nabsys, Inc. Devices and methods for determining the length of biopolymers and distances between probes bound thereto
JP5717634B2 (en) 2008-09-03 2015-05-13 ナブシス, インコーポレイテッド For voltage sensing of biomolecules and other analytes in the fluid channel, the use of nanoscale electrodes are displaced in the longitudinal direction
US9650668B2 (en) 2008-09-03 2017-05-16 Nabsys 2.0 Llc Use of longitudinally displaced nanoscale electrodes for voltage sensing of biomolecules and other analytes in fluidic channels
US8455260B2 (en) 2009-03-27 2013-06-04 Massachusetts Institute Of Technology Tagged-fragment map assembly
EP2426214A1 (en) * 2010-09-01 2012-03-07 Koninklijke Philips Electronics N.V. Method for amplifying nucleic acids
US8715933B2 (en) 2010-09-27 2014-05-06 Nabsys, Inc. Assay methods using nicking endonucleases
JP5998148B2 (en) 2010-11-16 2016-09-28 ナブシス 2.0 エルエルシー The method for sequencing of biomolecules by detecting the relative position of the hybridized probe
WO2012109574A3 (en) * 2011-02-11 2012-12-20 Nabsys, Inc. Assay methods using dna binding proteins
US9914966B1 (en) 2012-12-20 2018-03-13 Nabsys 2.0 Llc Apparatus and methods for analysis of biomolecules using high frequency alternating current excitation

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5547839A (en) * 1989-06-07 1996-08-20 Affymax Technologies N.V. Sequencing of surface immobilized polymers utilizing microflourescence detection
US5302509A (en) * 1989-08-14 1994-04-12 Beckman Instruments, Inc. Method for sequencing polynucleotides
CA2044616A1 (en) * 1989-10-26 1991-04-27 Pepi Ross Dna sequencing
WO1993005183A1 (en) * 1991-09-09 1993-03-18 Baylor College Of Medicine Method and device for rapid dna or rna sequencing determination by a base addition sequencing scheme
FR2703052B1 (en) * 1993-03-26 1995-06-02 Pasteur Institut New method for sequencing nucleic acids.
US5641658A (en) * 1994-08-03 1997-06-24 Mosaic Technologies, Inc. Method for performing amplification of nucleic acid with two primers bound to a single solid support
US5695934A (en) * 1994-10-13 1997-12-09 Lynx Therapeutics, Inc. Massively parallel sequencing of sorted polynucleotides
US6255475B1 (en) * 1995-01-31 2001-07-03 Marek Kwiatkowski Chain terminators, the use thereof for nucleic acid sequencing and synthesis and a method of their preparation

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0148184A2 *

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