EP1492888A2 - Analyse de melanges de fragments d'acide nucleique et l'expression genique - Google Patents

Analyse de melanges de fragments d'acide nucleique et l'expression genique

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Publication number
EP1492888A2
EP1492888A2 EP03742963A EP03742963A EP1492888A2 EP 1492888 A2 EP1492888 A2 EP 1492888A2 EP 03742963 A EP03742963 A EP 03742963A EP 03742963 A EP03742963 A EP 03742963A EP 1492888 A2 EP1492888 A2 EP 1492888A2
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EP
European Patent Office
Prior art keywords
nucleic acid
acid fragments
fragments
mixture
fragment
Prior art date
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EP03742963A
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German (de)
English (en)
Inventor
Achim Fischer
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Sygnis Pharma AG
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Axaron Bioscience AG
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Publication of EP1492888A2 publication Critical patent/EP1492888A2/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING 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
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C12Q1/683—Hybridisation assays for detection of mutation or polymorphism involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]

Definitions

  • the invention relates to a method for analyzing nucleic acid fragment mixtures and the use of the method for gene expression analysis.
  • sequenced expressed sequence tag
  • sequenced expressed sequence tag
  • the abundance of a transcript is no longer represented by the frequency of an event, for example the frequency with which a clone representing this transcript occurs, but by the intensity of the respective band.
  • This largely eliminates the redundancy that characterizes the EST sequencing of the prior art, which is associated with a cost reduction.
  • the respective bands are isolated from the gel, reamplified by means of PCR and cloned. More modern variants of this method, as are described, for example, in EP 0 743 367, are based on fragment generation by means of restriction digestion of double-stranded cDNA, which significantly increases the reproducibility of the fragment patterns obtained.
  • fragment-specific information such as fragment length, partial nucleotide sequence, information about position and / or orientation of the fragment within the starting cDNA etc.
  • fragment-specific information such as fragment length, partial nucleotide sequence, information about position and / or orientation of the fragment within the starting cDNA etc.
  • bp long partial sequence which is known for each fragment, as well as the information about the distance of this sequence from the 3 'end of the fragment.
  • the method described has disadvantages which lead to the unreliability of said signatures: (1) the identification of 4 nucleotides of the 8 bp long sequence, which is carried out by “invasive” or “selective” amplification primers, is imprecise, since primers are often also incorporated whose selective portion, namely the nucleotides located at the 3 'end, are not perfectly complementary to the template, and (2) the determination of the fragment length via the electrophoretic mobility is inaccurate, since the mobility of a fragment in addition to the length of G / C content and the exact sequence of the fragment (cf. Forensic Sei. Int.
  • the fragments obtained are separated by gel electrophoresis , its length and therefrom the distance between the two restriction endonuclease recognition sites on which the formation of a fragment is based, and signatures are generated, consisting of the sequence of the first recognition site, the sequence of the second recognition site and the assumed distance between the two recognition sites (expressed in base pairs) Using these signatures, database searches are carried out in order to allocate the detected fragments to the genes from which the fragments are derived, which also shows that due to great uncertainties in the determination of fragment sizes on the basis of fragment mobilities, there is a high response Part of incorrect assignments of database entries to detected fragments occurs.
  • the object of the present invention is achieved by a method for analyzing nucleic acid fragments, comprising the steps:
  • step (b) incubation of at least a subset of the mixture of nucleic acid fragments from step (a) with at least one restriction endonuclease, the interface of which lies outside of its recognition site, (c) identification of one or more nucleotides of the cut nucleic acid fragments from (b), the identification being simultaneous for several or all nucleic acid fragments are carried out.
  • the object according to the invention is achieved by a method for analyzing nucleic acid fragments, comprising the steps:
  • step (a) Provision of a mixture of nucleic acid fragments which have at least one recognition site for a restriction endonuclease cutting outside their recognition site, (b) incubation of at least a subset of the mixture of nucleic acid fragments from step (a) with at least one restriction endonuclease, the interface of which lies outside of its recognition site and which creates overhanging ends of known position and length but unknown sequence,
  • the mixture of nucleic acid fragments is preferably, optionally amplified, restriction fragments of cDNA or genomic DNA.
  • the fragments or a part of the fragments can be flanked by sequence regions common to all or some fragments. These common sequence regions can be, for example, linkers or adapters attached to the fragments, that is to say double-stranded nucleic acid fragments which can be obtained by Hybridization of two essentially or at least partially complementary oligonucleotides are readily available.
  • adapters are characterized by a length between 5 and 200 nucleotides, preferably between 10 and 80 nucleotides, particularly preferably between 15 and 40 nucleotides.
  • the fragments preferably have a characteristic size distribution with a smallest occurring size, a largest occurring size and an average size, the size being influenced or determined by the positions and / or the frequency of the recognition site or sites for the restriction endonuclease or restriction endonucleases used for fragment generation of course, the length of any attached linker or adapter must also be taken into account.
  • a mixture of nucleic acid fragments preferably double-stranded cDNA, is cut with at least one restriction endonuclease, which preferably has a four-based recognition sequence.
  • restriction endonucleases are Alul, Bfal, Bst I, Chal, Csp6l, Cv JI, Cv / JI, Dpnl, Dpnll, Haelll, Hhal, HwPlI, Hpall, HpyCm IV, HpyCIl4 V, Mbol, Msel, Mspls, Nlalll , Sau3al, Tail, Taql, Tsp5091.
  • linker molecules are attached to one or both ends of the fragments thus obtained - generally via enzymatic ligation.
  • fragment ends and linker ends are compatible with one another, ie are smooth or have overhangs that are complementary to one another.
  • single-stranded fragment ends can be removed using a nuclease or, in the case of 5 'overhangs, filled in using a polymerase and thus converted into smooth ends if the attachment of linkers with smooth ends is intended.
  • Another example of post-treatment of fragment ends is partial filling, which can prevent, usually undesirable, ligation of two fragment ends to one another.
  • a palindromic and thus self-complementary overhang of the sequence 3'-CTAG-5 'generated by treatment with the restriction endonuclease Sau3al can be converted into a no longer self-complementary overhang of the sequence 5'-TAG-3' by treatment with a polymerase in the presence of dGTP , Only such linkers with an overhang 5 '-ATC-3' complementary to it could now be attached to such an overhang; ligation of two fragment ends with one another would no longer be possible.
  • an amplification with one or more PCR primers directed against the attached linkers or with one or more against the fragments is optionally carried out to display a desired subset of fragments attached linker directed PCR primer and in addition a PCR primer which is directed against a terminal region of the original nucleic acid fragments, preferably the starting cDNA molecules.
  • a PCR primer which is directed against a terminal region of the original nucleic acid fragments, preferably the starting cDNA molecules.
  • the region which was introduced by the cDNA primer used for cDNA synthesis or a region which is artificially attached to the 5 'end of the mRNA used for cDNA synthesis or to the 3' end of the first strand cDNA was added.
  • the cDNA primer used is preferably an oligo-dT primer which can have an extension by one or more nucleotides at its 3 'end and / or at its 5' end, at least some of which have no " If two or more restriction endonucleases which generate different ends are used for the fragment generation, different linkers can be used in the subsequent step, some of which attach to one type of end and another part to another type of end If these linkers differ not only in their ends and thus in their compatibility (ie, their attachability) to the fragment ends, but also in their remaining sequence, then in a subsequent PCR amplification, specific fragments can be targeted by suitable choice of the primers (those on the linker sequences of the selected primers under the set amplification conditions can be amplified), while certain other fragments (those to the linker sequences of which the selected primers cannot bind) remain unamplified.
  • a further possibility for the selective amplification of certain fragments is to use invasive primers which are extended at their 3 'end to the linker sequence common to all fragments by one or more additional selective bases (see for example EP 0 743 367).
  • a further possibility for selective isolation or amplification, which can be used in the course of the method according to the invention, is described in WO 94/01582.
  • Restriction endonucleases that cut outside their recognition site are those restriction endonucleases in which the partial sequence that triggers the enzyme activity (the recognition site), which is usually one of 4-8
  • Base pair of existing region of double-stranded DNA and on which the Enzyme binds to the DNA double strand, and the interface, that is to say the region of the DNA double strand in which the sugar phosphate backbone of the DNA strands is hydrolytically separated, is offset from one another on at least one of the two strands forming the double strand.
  • restriction endonucleases such as Fokl [cutting characteristic GGATG (9/13): the “upper” strand is cut 9 bases away from the recognition site GGATG, the “lower” strand 13 bases away from the recognition site] or Btsl [Cutting characteristic GCAGTG (2/0)] or the restriction endonuclease Bcgl [cutting characteristic (10/12) CGANNNNNNTGC (12/10): both strands are cut once in front of and once behind the recognition site].
  • Fokl cutting characteristic GGATG (9/13): the “upper” strand is cut 9 bases away from the recognition site GGATG, the “lower” strand 13 bases away from the recognition site] or Btsl [Cutting characteristic GCAGTG (2/0)] or the restriction endonuclease Bcgl [cutting characteristic (10/12) CGANNNNNNTGC (12/10): both strands are cut once in front of and once behind the recognition site].
  • restriction endonucleases Aarl, Acelll, Alol, Alw ⁇ , Bael, Bbr7l, Bbsl, Bbvl, BceA ⁇ , Beeil, BciYl, BfuAl, Bmrl, Bpli, Bpml, BpuEl, Bsal, BsaXl, BscAl, BseKIll, BseMIll , BsmBl, BsmFl, Bsp24l, BspCN I, BspMl, BsrOl, Bst ⁇ 5l, Cjel, CjeVl, Earl, Ecil, Eco57l, Eco57Ml, Fall, Faul, HaelV, Hgal, HinAl, Hphl, Mboll, Mmel, Mnll, Plel, Ppil PsrI, RleAl, Sapl, SfaNl, Sthl32l, Stsl, Taqll, TspOT I
  • restriction endonucleases which produce single-stranded overhangs, which can be both 3 'overhangs and 5' overhangs. If restriction endonucleases are to be used which produce smooth ends (for example Mlyl, cutting characteristic GAGTC (5/5), or SspO5 I, GGTGA (8/8)), the smooth ends can be converted into overhanging ends in an additional step.
  • T4 DNA polymerase incubation with T4 DNA polymerase in the presence of a selected nucleotide triphosphate; the exonuclease activity of the T4 DNA polymerase then degrades one of the two strands in the 3 '-' 5 'direction until the first "nucleotide of the same name" in the strand is reached (for example up to the first "G” if the nucleotide triphosphate dGTP used see Ausubel et al., Current Protocols in Molecular Biology (1999), John Wiley & Sons).
  • Another type of restriction endonucleases that cut outside their recognition site are enzymes in which the recognition site is interrupted by a sequence of any or largely any nucleotides.
  • enzymes such as Xcml (cutting characteristic CCANNNNN / NNNNTGG) or S / ZI (cutting characteristic GGCCNNNN / NGGCC).
  • Another special case to be considered outside of their recognition site of restriction endonucleases which are cutting is so-called "nicking endonucleases" which only cut one strand of a nucleic acid double strand.
  • Examples of such endonucleases are N.AlwI (GGATCNNN N) and N.BstNBI (GAGTCNNNN / N) the sense strand on the marked with "/" Cut position.
  • the recognition site for a restriction endonuclease that cuts outside of its recognition site in the nucleic acid fragments from the fragment mixture in (a) is preferably located within the terminal sequence regions common to many or all fragments of the mixture, in particular in the sequence regions of the adapters or linkers attached to the fragments.
  • the selection of the enzyme and the position of the recognition site are to be selected such that, when the restriction endonuclease or restriction endonucleases act, a "proximal" cut is made and the respective nucleic acid fragment is cut in the fragment-specific area which is outside the flanking linker common to all or many fragments
  • recognition sites for the restriction endonucleases to be used which are outside individual fragments and which lie outside the flanking linker areas common to all or many fragments are protected against recognition by the corresponding restriction endonuclease
  • recognition sites for certain restriction endonucleases can be achieved, for example, by incorporating methylated nucleotides such as methyl dCTP Protection against restriction endonucleolytic cleavage is also provided by using a methylase belonging to the selected restriction endonuclease.
  • the enzyme BamRl methylase converts recognition sites of the restriction endonuclease BamHl into their C-methylated form, which Bam ⁇ l no longer recognizes and cuts.
  • the enzyme CpG methylase methylates CG dinucleotides, for example preventing a cut of a DNA fragment containing the sequence CGTCTC with the restriction endonuclease BsmBl (cutting characteristic CGTCTC (1/5)).
  • the above measures ensure that each nucleic acid fragment present in the mixture in the course of a restriction induction only on exactly one predetermined position is cut.
  • nucleic acid molecules preferably cDNA or genomic DNA
  • restriction endonuclease from step (b)
  • linker molecules to the ends generated with the latter and to carry out a PCR amplification with primers which are directed against the terminal linker molecules.
  • One or more nucleotides of the cut nucleic acid fragments can be identified in several different ways. In particular, three preferred procedures are suitable for this, which should not, however, preclude further procedures:
  • the terminating nucleotides carry marking groups which can be used to detect incorporation
  • the four dideoxynucleotides carry four distinguishable labeling groups, in particular four different fluorophores, and it can then be recognized from the fluorescence activity which of the four
  • restriction endonucleases are particularly suitable: Aarl, Acelll, Alwl, Bbr7l, Bbsl, Bbvl, BceAl, Bce, BfuAl, Bsal, BscAl, BsmAl, BsmBl, Bsm ⁇ l, BspMl, Earl, Faul, Fokl, Hgä, Plel, S SfaNl, SM321, Stsl. Attachment of adapters with overhanging ends of suitable length and suitable type (3 'overhang or 5' overhang) to fragments having an overhanging end, the attachment being carried out in a sequence-specific manner.
  • the overhanging fragment ends can in particular have been generated using one of the following restriction endonucleases: Aarl, Acelll, Alol, Alwl, Bael, Bbr7l, Bbsl, Bbvl, BceAl, Bcett, Bcgl, BciVl, BfuAl, Bmrl, BpR, Bpml, BpuEl,
  • Sequencing adapter used, which differ in their overhang.
  • a sequential or a parallel procedure is of course also conceivable, in which different adapters are used in separate fastening reactions.
  • adapters bearing marking groups which differ both in their overhang and in their marking group.
  • the labeling groups are fluorophores, so that the fluorescence activity of the fastening products shows which adapter has been fastened to a given fragment end.
  • Mixture can be, for example, adapters of the general structure
  • adapter means the double-stranded portion of the adapter
  • X represents one of the four possible nucleotides in the form of a single-stranded overhang
  • F means a fluorophore which identifies the overhanging base X.
  • N is a mixture of all four possible nucleotides or a universal nucleotide such as inosine.
  • the first nucleotide of the dibasic fragment overhang would be determined by attaching the first adapter
  • the second nucleotide of the dibasic fragment overhang would be determined by attaching the second adapter.
  • the sequence of the overhanging end can be determined.
  • overhangs of a length of more than two nucleotides can also be sequenced.
  • marking groups which allow the simultaneous detection of more than four (namely usually an integer multiple of four) different markings.
  • the first four of the different markings could be used to identify a first base of fragment overhangs generated, the second four of the different markings for identification of a second base of the fragment overhangs generated, and possibly further sets of four different markings for further bases of the fragment overhangs generated , Such a “multiplexing” would result in a reduction in the number of experimental steps required.
  • so-called “quantum dots” could be considered as marker groups, of which numerous different ones can be detected in a common measurement without the measurement results influencing one another (Han et al., Nat. Biotechnol. 19, 631-5 [2001]).
  • Extension of selective oligonucleotide primers whose nucleotide or nucleotides located at the 3 'end can hybridize with the nucleotide or nucleotides to be sequenced, followed by the identification of those primers which were extended in the extension reaction. If necessary, the extension can be carried out by means of the polymerase chain reaction (PCR).
  • Linkers or adapters are preferably attached to the ends of the nucleic acid fragments to be sequenced, which can serve as primer binding sites common to all or many fragments.
  • the oligonucleotide primers are then designed in such a way that, after denaturing the nucleic acid fragments to be sequenced, they correspond to those at the 3 'end of the
  • Nucleic acid fragment strands can hybridize attached linker strand. It must be ensured here that the oligonucleotide primers hybridized in this way "overlap" by one or more nucleotides with the region of the nucleic acid fragment adjacent to the linker region, that is to say at their 3 'end they have nucleotides which correspond to the nucleotides of the
  • nucleic acid fragments if there is complementarity.
  • selective nucleotides which allow the primer to be extended by means of a polymerase if they have become part of a double strand as a result of said hybridization, but which at least largely prevent the primer from being extended if they do not
  • nucleotides are identified simultaneously for several or all nucleic acid fragments, preferably after the nucleic acid fragments contained in the mixture have been separated according to a fragment-specific property, in particular according to the size and / or mobility of the fragments, by electrophoretic separation.
  • the method of gel electrophoresis is particularly preferred, in which flat gels or gel-filled capillaries are used for the separation.
  • enzymatic reactions according to variants 1-3 are carried out in step (c) in such a way that one or two nucleotides of the fragments are identified in parallel batches, the nucleotides of the fragments to be identified in the parallel batches being in a defined position relative to one another , for example adjacent to each other.
  • One or two nucleotides of known position are then first determined in parallel separations of the said approaches for each of the separated fragments, which is preferably done by means of different marker groups which allow information about the nucleotides to be determined.
  • the determined nucleotides for individual or all of the separated fragments are then brought into the order in which they are present on the associated starting fragment from the mixture of nucleic acid fragments.
  • signatures in the form of short sequence sections which characterize the associated fragment are generated for the examined fragments.
  • the length of these sequence sections is preferably at least 14 bases, more preferably at least 16 bases, in particular at least 20 bases.
  • a signature can also contain other information characterizing a fragment, for example exact or approximate distances (specified in base pairs) between characteristic areas of the fragment, for example the distance between two known sequence sections estimated with the aid of an internal length standard based on the electrophoretic mobility, between a known sequence section and a fragment end, or between both fragment ends.
  • the length of the sequence sections is preferably at least 10 bases.
  • the information content of a signature is preferably large enough to allow the unambiguous identification and / or isolation of the associated fragment.
  • experience has shown that approximately 14-20 base pairs of sequence information without additional information about distances within the fragment in question are usually sufficient to recognize a transcript containing this sequence segment from a mixture of cDNA molecules and to identify the associated gene.
  • t ⁇ g sequencing such as SAGE (Velculescu et al., Science 270: 484-487 [1995], WO 00/53806) or MPSS (Brenner et al., Nature Biotechnol.
  • the information content of a signature identifying a fragment can be increased, among other things, by the following information:
  • an additional statement about the fragment to be identified or the associated transcripts or genes in question could be, for example, "3 'fragment double-stranded cDNA generated by the restriction endonuclease Rsal", whereby the identity of the sequence part of a signature with a 5' ⁇ 3 'direction is "above” (upstream) or "Before" the sequence region of a transcript located most towards the 3 'end of the fragment would be recognized as insignificant.
  • signatures whose sequence portion was in the wrong orientation with regard to the 5' -3 'preferred direction of an mRNA Sequence or the cDNA sequence derived from it is not recognized as significant.
  • additional information is given as to which molecular biological procedure the signatures were generated, which precludes the occurrence of certain partial sequences as a signature or part of a signature about genes in question could, for example, be "from the totality of all genes expressed in the leaf” if transcripts from leaf samples are to be identified by means of plant signatures generated, but for example genes which are only expressed in the root should not be taken into account.
  • the simultaneous identification of one or more nucleotides of the cut nucleic acid fragments takes place in step c) via the following individual steps: ca) identification of a first nucleotide of the cut nucleic acid fragments from b), the identification being carried out simultaneously for several or all nucleic acid fragments, cb) optionally identifying a further nucleotide of the cut nucleic acid fragments from b), the identification being carried out simultaneously for several or all nucleic acid fragments, cc) optionally repeating step (c b) until the desired number of
  • Nucleotides has been identified, cd) summarizing the sequence information obtained in steps (ca) to (cc) for a selected group or all nucleic acid fragments into fragment-specific signatures, a signature being able to contain further information about the respective fragment in addition to the sequence information, wherein the nucleotide identification in steps ca) to cc) optionally also includes the separation of the nucleic acid fragments of the mixture.
  • step a) At least a subset of the mixture of nucleic acid fragments provided in step a) is subjected to the following process steps aa) to ad):
  • aa separation of the mixture of nucleic acid fragments according to at least one fragment-specific property, ab) optionally detection of the relative frequency of some or all fragments in the separated mixture, ac) optionally comparing the information obtained in (aa) and / or (ab) about the composition of different mixtures of nucleic acid fragments according to step (a), ad) optionally registering nucleic acid detected in (ab) and / or (ac) Fragments which occur in different mixtures of nucleic acid fragments at different relative frequencies ⁇
  • III) is a mixture of nucleic acid fragments which is at least partially identical to I) or II).
  • At least one fragment of one of groups (I) to (III) is obtained in an additional process step.
  • the fragments of interest are preferably obtained by specific PCR amplification from a mixture of nucleic acid fragments, in which fragment-specific oligonucleotide primers are used, which are accessible and can be produced by the signatures determined in step (cd).
  • a further preferred embodiment relates to one of the above methods according to the invention, in which a mixture of nucleic acid fragments according to step a) or a subset of this mixture of nucleic acid fragments according to step a) is provided, which was produced by the following steps: i) flanking on both sides of the restriction fragments of the mixture with identical or different adapters ii) hybridizing the fragments from step (i) with different primers in each case, all of which have regions complementary to the adapters from step (i) and which have their respective 3 'ends have one or more nucleotides, which after
  • Hybridization of the primer with its target sequence protrude beyond the region complementary to the adapter and are complementary to the nucleotides opposite them in the double strand of a subset of the fragments from the nucleic acid mixture from (a). iii) sequence-specific extension of the primers from ii) and, if appropriate, subsequent PCR amplification of the nucleic acid fragments from the fragment mixture which had been extended in a sequence-specific manner in step ii).
  • Sequence-specific extension means that only those primers whose nucleotide or nucleotides at the 3 'end at step 3) are or are complementary to the opposite nucleotides of the fragment with which they hybridize a nucleic acid are hybridized - Have formed double strand.
  • a method for gene expression analysis comprising the following steps:
  • fragments of interest optionally obtained fragments of interest from the mixture of nucleic acid fragments from (al) or (b1), the fragments of interest being the fragments registered in (e), ml) optionally identifying the genes belonging to the nucleic acid fragments of interest, from which the nucleic acid fragments are derived by means of
  • the fragments of interest can be the fragments registered in (e).
  • steps (fl) to (i) are repeated, changing the position and / or sequence of the recognition site ensures that nucleotide positions of the fragments to be analyzed other than those previously examined are converted into single-stranded overhangs and thus further, previously unidentified Nucleotides can be identified.
  • a simultaneous approach in parallel approaches is of course also possible.
  • the procedure is as follows: At least one fragment mixture is provided in which many or all fragments have identical ends, for example smooth ends or overhanging ends of the same length and sequence. This mixture is aliquoted, for example in 10 substantially equal aliquots.
  • Each of the mixtures is mixed with one of a selection of different adapters (here one of 10 different adapters) and exposed to ligation conditions, with all adapters being distinguished by an end that is compatible with the fragment ends, that is to say attachable to them. Furthermore, all adapters have at least one recognition site for a restriction endonuclease that cuts outside their recognition sequence, for example Mmel.
  • the adapters differ from one another in that the recognition sequence is removed from the adapter end to be attached to the fragment ends.
  • two different adapters differ in this distance by an integer multiple of the length of the overhanging ends, which can be generated by said restriction endonuclease which cuts outside their recognition sequence.
  • the distance is accordingly 18 bp in some adapters, 16 bp in other adapters, 14 bp, 12 bp, 10 bp, 8 bp, 6 bp, 4 in the other adapters bp, 2 bp or 0 bp.
  • bases 19 and 20 are in the case of the first reaction, bases 17 and 18 in the second reaction, and bases 15 and 16, 13 in the remaining reactions and 14, 11 and 12, 9 and 10, 7 and 8, 5 and 6, 3 and 4 or 1 and 2 in the form of a single-stranded overhang.
  • the complete set of all 10 reactions thus allows the identification of a coherent, 20 base long partial sequence or signature for the fragments present in the fragment mixture.
  • recognition sites for restriction endonucleases cutting at different distances from their recognition sites at the same position.
  • an adapter could have one at its end to be attached to the fragment ends Detection point for Earl (cutting characteristic CTCTTC (l / 4)), a second adapter in the same position a detection point for SfaNl (cutting characteristic GCATC (5/9)) and a third adapter in the same position a detection point for Stsl (cutting characteristic GGATG (10/14 )), which made it possible to use the method according to the invention to identify 13 base partial sequences of the fragments.
  • Detection point for Earl cutting characteristic CTCTTC (l / 4)
  • a second adapter in the same position a detection point for SfaNl (cutting characteristic GCATC (5/9)
  • a third adapter in the same position a detection point for Stsl (cutting characteristic GGATG (10/14 )
  • a method for gene expression analysis comprising the following steps:
  • a2) provision of at least one mixture of nucleic acid fragments, in particular a mixture of cDNA fragments, having at least one recognition site for a restriction endonuclease that cuts outside of its recognition site and that is located on linkers attached to starting fragments, b2) incubation of the mixture of nucleic acid fragments from ( a2) with the restriction endonuclease or the restriction endonucleases from step (a2), c2) identification of a first nucleotide of the cut nucleic acid fragments from (b2), the identification being cut simultaneously for several or all nucleic acid fragments of the mixture and with separation of the mixture and for
  • Identification of the nucleotide of suitably treated nucleic acid fragments takes place according to at least one fragment-specific property, d2) optionally identification of a further nucleotide of the cut nucleic acid fragments from (b2) according to (c2), e2) optionally repetition of step (d2) until the desired number of Nucleotides has been identified, f2) optionally repeating steps (a2) to (e2) one or more times, the position and / or sequence of the recognition site being changed in such a way that the repetition or repetitions in each case the identification of previously unidentified nucleotides allow, g2) combining the sequence information obtained in steps (c2) to (f2) for a selected group or all nucleic acid fragments into fragment-specific ones Signatures, where a signature can contain further information about the respective fragment in addition to the sequence information, h2) assignment of the fragment-specific information obtained in the separation according to a fragment-specific property in (c2) to the signatures obtained in (g2) for the nucleic acid fragments, being the
  • information includes the relative or absolute mobility of the fragments and / or the apparent or actual fragment length determined on the basis of a length standard and the assignment can take place in tabular and / or computer-readable form, i2) if necessary, identification of the nucleic acid Fragments belonging to genes from which the nucleic acid fragments derive, by searching electronic databases for the signatures from (g2), j2) optionally providing at least one further mixture of nucleic acid fragments, in particular a mixture of cDNA fragments, obtained from analogue ones Way to the mixture of nucleic acid fragments from (a2), it being possible here to dispense with the addition of linkers having at least one recognition site for a restriction endonuclease that cuts outside its recognition site, k2) separation of the mixture or mixture the nucleic acid fragments from (i2) according to a fragment-specific property essentially under the conditions of the separation into (c2),
  • Separation of the fragments may include the relative or absolute mobility of the fragments and or the apparent or actual fragment length determined on the basis of a length standard and the assignment may be in tabular and / or computer-readable form, m2) if necessary, comparison of the relative or absolute frequencies of at least part of the fragments separated in (k2) with the relative or absolute frequencies of the respective homologous, that is to say completely or essentially sequence-identical fragments originating from different mixtures of nucleic acid fragments, n2) where appropriate, registration of those fragments whose relative or absolute frequency differ from the relative or absolute frequency of their homologous fragments of other mixtures of nucleic acid fragments by at least a preselected factor, o2) where appropriate, assigning the fragments registered in (n2) to those genes or Transcripts, from which said fragments are derived, using the results obtained in step (h2), p2) optionally obtaining the fragments registered in (n2) from the mixture of nucleic acid fragments from (a2) or (i2) and / or (j
  • steps (i2) to (n2) can also be carried out before steps (a2) to (h2).
  • nucleic acid fragments preferably mixtures of cDNA fragments
  • Mixtures of nucleic acid fragments can be produced by methods known from the prior art.
  • EP 0 743 describes
  • Fragment subpools obtained from RNA preparations are then separated by gel electrophoresis according to their size, and the bands obtained. Signal patterns are compared with each other. Bands or signals originating from homologous fragments, the intensity of which differs between different samples, represent genes that are in the compared ones
  • the fragment-specific property is a property, in particular a physical or physicochemical property, which can be realized by different molecules within a continuum or in the form of a larger number (for example at least 10 or at least 100) of different gradations or forms. It is particularly preferred to use different mobility of different nucleic acid fragments in separation systems, in particular different electrophoretic mobility in electrophoresis systems such as agarose or polyacrylamide gel electrophoresis. Mobility is usually influenced by the length of a fragment; however, it is not a strictly linear relationship, since the G / C content and conformation of a nucleic acid molecule also influence mobility. Therefore, the mobility of a nucleic acid molecule can usually only be used for approximate, but not for absolute size determination.
  • the fragment-specific property can be a specific partial sequence of n nucleotides, where n can be equal to or greater than 1.
  • Said partial sequence of a fragment preferably adjoins a linker attached to the end of the fragment, so that a mixture of different fragments according to this partial sequence can be separated by extension, optionally a repeated extension in the form of an amplification, of selective oligonucleotide primers.
  • extension optionally a repeated extension in the form of an amplification, of selective oligonucleotide primers.
  • separation of a fragment mixture means the production of mixtures of amplified fragments, each of which copies produced by amplification contains only a part of the fragments present in the starting mixture.
  • said partial sequence is at least partly in the form of a single-stranded one Overhang, and a mixture of different fragments after this partial sequence is separated by attaching adapters with compatible overhangs.
  • This process also called “categorization of nucleotide sequence populations”, is described in WO 94/01582. A combination of both measures is also conceivable and is described, for example, in WO 01/75180.
  • the relative frequency of some or all of the fragments is detected by measuring the signal strength obtained in the detection of individual nucleic acid fragments.
  • the nucleic acid fragments contain detectable labeling groups, the use of fluorophores as labeling groups being particularly preferred.
  • the relative frequency of a fragment as an area under the corresponding curve in the fluorogram (the plot of the measured fluorescence intensity as a function of the retention time) is readily available in the form of a numerical value.
  • a fragment here is to be understood as the entirety of all the sequence-identical nucleic acid molecules of a mixture, possibly plus the sequence of complementary nucleic acid molecules.
  • the numerical values obtained as the relative frequency of fragments are often stored in a computer-readable form.
  • nucleic acid fragments preferably cDNA fragments, of different relative frequencies
  • those fragments are identified which differ in their proportion between different biological samples or between different mixtures of cDNA fragments. If care is taken to generate cDNA fragments from the mRNA molecules present in the samples, the frequency distribution of which is similar or even equal to the frequency distribution of the different mRNA molecules, then cDNA fragments between mutually compared fragment mixtures of different frequencies also show mRNA -Molecules of different frequencies and thus differentially expressed genes.
  • a threshold value for frequency differences can optionally be set, so that, for example, only those cDNA fragments whose relative frequency between mutually compared fragment mixtures is at least the same are further investigated Factor two differs.
  • the simultaneous identification of a nucleotide or a plurality of nucleotides for several or all nucleic acid fragments is preferably carried out by, as described above, on the overhanging fragment ends generated by means of at least one restriction endonuclease cutting outside their recognition site for identity the characteristic process of the nucleotide to be identified is carried out, for which a mixture of several or all nucleic acid fragments is used and the result of which can preferably be observed by incorporating a label, in particular a fluorescent label.
  • the identified nucleotides lie in proximity to one another, that is to say that the information about the nucleotide identities thus obtained results in a coherent partial sequence of the respective nucleic acid fragment.
  • the “sequencing reaction” has expired, the products formed in the process are separated, in which case the separation can again take place according to the fragment-specific property from (b1) or (c2).
  • the nucleotide identity obtained for some positions is assigned to each or some of the separated nucleic acid molecules.
  • the information received about a fragment is called a signature.
  • the signature can also contain further information, for example sequence information obtained in another way or approximate fragment size obtained via fragment mobility.
  • fragment-specific signatures can be determined for all or part of the fragments contained in a fragment mixture.
  • signatures are determined in particular for those fragments which differ in their relative frequency between the fragment mixtures to be compared by at least one fixed factor. Otherwise, the sequence portion of a signature does not necessarily have to be a coherent sequence.
  • partial nucleotide sequences of both fragment ends of a given fragment are determined and combined to form a signature; it is of course also possible to include additional information in the signature, such as approximate fragment lengths.
  • the signature could be for a particular fragment
  • the fragment "begins” with the nucleotide sequence CTCA at the 5 'end, "ends” with the nucleotide sequence GGAT at the 3' end and in total, possibly plus terminal linker regions, approximately 200 bp ( 4 bp + 192 bp + 4 bp) is long.
  • the formulation "approximately” takes into account that the length determination of fragments based on the electrophoretic mobility, as stated above, is subject to a certain error.
  • Fragments of interest can be obtained from the mixture of nucleic acid fragments, preferably cDNA fragments, with the aid of the fragment-specific signatures determined, for example by means of PCR using gene-specific primers.
  • a mixture of 3'-cDNA fragments was obtained by means of the restriction endonuclease Rsal, followed by the ligation of linker to the (smooth) fragment ends, and if the above signature GTACAGTA was obtained for a selected fragment Fragment known that after the Rs 1 cut (removal of the first two nucleotides of the Rs ⁇ l recognition site, GT), the first nucleotides following the linker sequence are the sequence ACAGTA.
  • a primer is now used for PCR amplification which, following the linker sequence at its 3 'end, has precisely this nucleotide sequence ACAGTA
  • the associated fragment can be accessed directly by amplification from the fragment mixture, since said primer selectively promotes amplification of those fragments. with which it is sequence-identical (or complementary) over its entire length.
  • the fragment obtained in this way can then be subjected to a further analysis, for example sequencing, followed by a database query for entries which are identical or similar to sequences.
  • a prerequisite for this procedure is of course a sufficiently high information content of the signature, ie a sufficient length and thus specificity of the fragment-specific area of the amplification primer.
  • the primer used would therefore have to be extended at its 3 'end by further specific bases. It must also be taken into account here that the greater the distance from the 3 'end of the primer, the less the ability of polymerases to discriminate against the extension of primers hybridized with partial mismatch with the template strand are. If a primer is extended at its 3 'end to increase the specificity by further fragment-specific bases, a certain loss in the specificity of those bases which immediately follow the sequence section of the primer complementary to the respective linker sequence is to be expected.
  • the signatures obtained for nucleic acid fragments of interest are used to design fragment-specific oligonucleotide primers.
  • the genes belonging to the nucleic acid or cDNA fragments of interest can be identified by searching electronic databases if the information content of a signature is high enough to permit clear or largely unambiguous identification of a gene and if the database has corresponding entries. How high the information content of signatures of a biological species has to be in order to allow a clear assignment of a signature to the associated gene can be determined empirically and can even differ from gene to gene within a biological species; for example, a particular decamer (a 10-nucleotide signature) may be characteristic of a single gene, while another decamer appears in many different genes.
  • the signatures obtained for nucleic acid fragments of interest are used to identify the nucleic acid fragments in a database search.
  • the signatures obtained for nucleic acid fragments of interest are used to create EST banks.
  • the signatures obtained for the individual fragments obtained from a cDNA preparation are used in order to design fragment-specific oligonucleotide primers. These are then used to obtain the respective fragments by means of PCR amplification. The fragments obtained are finally sequenced and the sequences are recorded in a database.
  • EST banks generated in this way can also be referred to as normalized EST banks, since each fragment is generated only once, regardless of its abundance or the abundance of the mRNA or cDNA molecules it represents.
  • redundancy can be practically excluded; however, in contrast to normalized banks according to the prior art, the redundancy information of the individual fragments, clones and those transcripts from which they are derived is not lost. Rather, abundance information can be obtained from the respective separation, for example by means of capillary gel electrophoretic separation The signal strength of the individual fragments of an examined fragment mixture can be taken and added to each received EST sequence as additional information.
  • mixtures of genomic DNA or cDNA generated restriction fragments flanked on both sides by identical or different adapters are used as a mixture of nucleic acid fragments, the fragments flanked by the adapter first being amplified by means of their 3 'end via the complementary one to the adapter Be subjected to a range extended by one or more nucleotides of primers and the amplification products thus obtained are used to carry out the method.
  • fragments are used as a mixture of nucleic acid fragments which were generated by restriction digestion with at least some of the type IIs restriction endonucleases from genomic or cDNA and which are flanked on one or both sides by adapter sequences.
  • overhanging ends are produced from the type IIs restriction endonucleases used, the sequence of which is not determined directly by the restriction endonuclease, but by the nucleic acid sequence of the interface and which can consequently differ from fragment to fragment.
  • adapters can be used for attachment which can only be attached to certain overhanging ends, in particular those whose nucleotide sequence is complementary to the nucleotide sequence of the overhanging adapter ends.
  • the required enzymatic reaction batches are created using an automatic pipetting device.
  • the fluorograms obtained by means of gel electrophoresis are automatically evaluated.
  • a Signals of different fluorograms belonging to one another are assigned to the computer system, which (i) homologous fragments from different mixtures of nucleic acid fragments, (ii) fragments of a nucleic acid mixture and the reaction products which were obtained for the identification of one or more nucleotides of the fragments of this mixture, (iii) Reaction products, which were obtained for the identification of several nucleotides of the fragments of a mixture of nucleic acid fragments, represent.
  • Such an automatic assignment can take place, for example, according to the following instruction:
  • Fragment length and signal intensity of all previously assigned signals is compared, 4. Cancel the process if the differences from (2) a preselected one
  • the automatic evaluation carry out the steps (dl), (el), (gl), (hl), (il), oil), (kl), (ml), (c2), (d2) , (e2), (f2), (g2), (h2), (i2), (12), (m2), (n2) and / or (o2).
  • Fig. 4 the identification of a nucleotide for all fragments of a mixture of nucleic acid fragments
  • Fig. 5 the identification of four nucleotides for all fragments of a mixture of nucleic acid fragments.
  • Figure 2 shows sequencing of the second position of the overhanging ends. Sequencing of a nucleic acid fragment representing the 3 'end of a cDNA molecule is shown.
  • the one for sequencing The adapters used are distinguished by a different sequence of the overhanging ends and by different marker groups which code for the sequence of the respective overhanging end.
  • a marker group indicating the base A is indicated by a dotted adapter, a marker indicating a C by a hatched adapter, a marker indicating a G by a filled adapter and a marker indicating a T by a cross-hatched adapter.
  • a marker group attached to the fragment in (1) by ligation and indicating a T indicates that the first base of the overhang is the complementary base A.
  • a labeling group attached to the fragment in (2) by ligation and indicating a C indicates that the second base of the overhang is the base G complementary thereto.
  • FIG. 3 shows the generation of various overhanging ends by shortening a nucleic acid fragment
  • FIG. 3 shows the release of shortened overhanging fragment ends which, with respect to the double-stranded region of the starting fragment, positions -5 and -6 (left), -3 and -4 (middle) and -1 and -2 (right) in terminal single-stranded and thus contain sequencing accessible via adapter ligation.
  • a 3 'cDNA fragment obtained by means of the restriction endonuclease Mbol is shown here as the starting fragment.
  • Figure 5 shows the identification of four nucleotides for all fragments of a mixture of nucleic acid fragments (fragments 1-7).
  • sequence signatures result:
  • FIG. 6 shows the separation of a mixture of nucleic acid fragments by means of capillary gel electrophoresis.
  • cDNA fragments were generated from a suspension culture of Saccharomyces cerevisiae.
  • the signals obtained from a stationary phase (gray) and from a culture in the logarithmic phase (black) are shown.
  • the horizontal scale shows the fragment size, the vertical scale shows the fluorescence intensity.
  • E one of the fragments from a mixture of nucleic acid fragments, B1-B16, identification of the first to sixteenth base of the fragment, FAM, PET, VIC, NED, the respective fluorophore detected during the identification of a base, (G), (A ), (T), (C), the base identified by means of the respective fluorophore.
  • FAM the first to sixteenth base of the fragment
  • FAM the respective fluorophore detected during the identification of a base
  • G A
  • T the base identified by means of the respective fluorophore.
  • the bar at the top shows the fragment size, so it is a fragment with a size of approximately 140 bp.
  • FIG. 8 shows a list of some signatures obtained from a suspension culture of Saccharomyces cerevisiae.
  • the fragment size, the signatures determined according to the method according to the invention, the open reading frames (ORFs) identified by means of BLAST analysis and the signal intensity obtained by means of capillary gel electrophoresis are to be specified in each case.
  • FIG. 9 shows the identification of several nucleotides of four nucleic acid fragments of a mixture of nucleic acid fragments.
  • the fragments are approximately 75 bp, 77 bp, 78 bp and 79 bp in length.
  • F separated fragments of the mixture, B1-B6, identification of the first to sixth bases of the fragments, FAM, PET, VIC, NED, the respective fluorophore detected when identifying a base, (G), (A), T), ( C), the base identified by means of the respective fluorophore.
  • the signature TCATTG results for the fragment with a length of 75 bp, the signature ACTGGC for the fragment with a length of 77 bp, with the signature ATGCCT for the fragment with a length of 78 bp, and for the fragment with a length of 79 bp Signature TATGCT.
  • RNA from a suspension culture of Saccharomyces cerevisiae was precipitated with ethanol and dissolved in 15.5 ⁇ l of water.
  • 10 ⁇ M cDNA primer CP31V 5 -ACCTACGTGCAGATTTTTTTTTTTTTTTTTTTTV-3 ', SEQ ID NO: 1 were added, denatured for 5 minutes at 65 ° C. and placed on ice.
  • the mixture was mixed with 3 ul 100 mM dithiothreitol (Life Technologies GmbH, Düsseldorf), 6 ul 5x Superscript buffer (Life Technologies GmbH, Düsseldorf), 1.5 ul 10 mM dNTPs, 0.6 ul RNase inhibitor (40 U / ul ; Röche Molecular Biochemicals) and 1 ⁇ l Superscript II (200 U / ⁇ l, Life Technologies) and incubated at 42 ° C. for 1 hour for cDNA first strand synthesis.
  • the pellet was dissolved in a restriction mixture consisting of 15 ⁇ l 10X universal buffer, 1 ⁇ l Mbol and 84 ⁇ l H 2 O and the reaction was incubated at 37 ° C. for 1 hour. It was extracted with phenol, then with chloroform and precipitated with ethanol.
  • the pellet was prepared in a ligation mixture from 0.6 ⁇ l lOx ligation buffer (Röche Molecular Biochemicals), 1 ⁇ l 10 mM ATP (Röche Molecular Biochemicals), 1 ⁇ l linker ML2025 (produced by hybridization of oligonucleotides ML20 (5 -TCACATGCTAAGTCTCGCGA-3 ', SEQ ID NO: 2) and LM25 (5'-GATCTCGC GAGACTTAGCATGTGAC-3 ', SEQ ID NO: 3)), 6.9 ⁇ l H 2 O and 0.5 ⁇ l T4 DNA ligase (1 U / ⁇ l; Röche Molecular Biochemicals) dissolved and the ligation performed overnight at 16 ° C.
  • ML2025 produced by hybridization of oligonucleotides ML20 (5 -TCACATGCTAAGTCTCGCGA-3 ', SEQ ID NO: 2) and LM25 (5'-GATCTCGC GAGACTTAGCATGTGAC
  • the ligation reaction was made up to 100 ⁇ l with water, extracted with phenol, then with chloroform and, after addition of 1 ⁇ l glycogen (20 mg / ml, Röche Molecular Biochemicals), precipitated with 100 ⁇ l 28% 8000 polyethylene glycol (Promega) / 10 mM MgCl 2 , The pellet was washed with 70% ethanol and taken up in 40 ul water.
  • Example 2 Amplification of cDNA-3 'restriction fragments divided into subpools
  • PCR batches were prepared, containing 2 ⁇ l of the ligation reaction from example 1, 2 ⁇ l of 10 ⁇ PCR buffer (670 mM Tris-Cl, pH 8.8, 170 mM
  • Primer ML20 had a fluorescent label (selected from one of the dye sets 5'-FAM, 5 '-JOE, 5'-ROX and 5' -TAMRA [dye set 1] or 5'-FAM , 5'-VIC, 5'-NED and 5'-PET [dye set 2]; further processing of the samples according to Example 3), or ML20 was used unlabelled (further processing of the samples according to Example 4 or Example 5).
  • the fluorograms were displayed and evaluated using the GeneScan version 3.7 software for Windows NT (Applied Biosystems To identify differentially expressed genes, fluorograms obtained with RNA preparations from yeast cells in different growth stages, but with the same amplification primers of the first and the second round of amplification, were compared with one another.
  • the fluorograms were matched using GeneScan and visually detected differences in the For comparisons of this type, the GeneScan function "align data by size" was first used to ensure that "matching" fragments from RNA preparations of different growths (ie representing the same gene / transcript) were used stadiums could be assigned to each other. In the next step, the signal strengths were normalized by adapting the average height of the signals of a sample to the average signal strength of a sample to be compared therewith.
  • fragments of identical size and thus identical transcripts which occur in comparing samples, and whose intensities differ from one another after normalization by at least a preselected factor, including the signature determined, were tabulated; In some cases, the associated values for fragment length (determined using the internal length standard), the signal intensity and the information about the amplification primers used were also included. For general transcriptome analysis (ie an "inventory" of expressed genes), all the signatures determined were tabulated, regardless of relative signal strengths.
  • the pellets were taken up in 20 ⁇ l of a ligation batch, containing 1.2 ⁇ l lOx ligation buffer (Röche), 8 ⁇ l 0.5 ⁇ g / ⁇ l Eco57I linker and a linker selected from ECO 1/2 to ECO11 / 12; see. Table 1; Production of the linkers by hybridization of the respectively stated complementary oligonucleotides) and 1 ⁇ l T4 DNA ligase (1 U / ⁇ l, Röche). It was ligated at 16 ° C overnight.
  • Example 3 a fragment occurring in Example 3 and the associated products from Example 4 shortened by means of Eco57l and provided with a sequencing adapter) were assigned to one another and recorded in tabular form, with the respective fluorophore also being used to determine the respective one Base identity was identified.
  • Such a table can, for example, have the format given in Table 3.
  • the partial cDNA sequences (“signatures”) obtained in this way were used to identify the relevant genes for a BLAST search.
  • the cDNA signature GATCTAGACAACCAAA which can be seen in Table 3, was used to code the yeast gene KTR4 (ORF YBR199W), coding for a putative Alpha-1,2-mannosyltransferase, Figure 8 shows further examples of signatures obtained from yeast.
  • the numerical values of this example relate to the use of Eco57l, which generates two-base overhangs for the identification of two neighboring bases ("doublets"), and of sequencing adapters, which optionally identify the first or the second base of such an overhang.
  • doublets the recognition sites for Eco57I in the Eco57I linkers are each offset by two bases
  • reaction according to example 3 results from the known recognition site of Mbol (cf. example 1)
  • Example 5 Determination of terminal bases via ⁇ // - / «reaction
  • a linker was selected from BCE1 to BCE13; see. Table 1; Production of the linkers by hybridization of the respectively stated complementary oligonucleotides) and 2 ⁇ l of Quick T4 DNA ligase (New England Biolabs) dissolved and ligated for 1 h at room temperature.
  • 2 ⁇ l of the ligation with 2 ⁇ l of 10 ⁇ M amplification primer 2 (identical in sequence to that strand of the Bce AI linker whose 3 ′ end had been linked to the fragments cut with Mbol), 2 ⁇ l of 10 ⁇ M CP31, 5 ul 10X Advantage 2 buffer, 1 ul 10mM dNTPs, 37 ul water and 1 ul 50x Advantage 2 DNA Polymerase Mix were mixed and amplified under the following conditions: 2 min. 94 ° C initial denaturation, then 25 cycles consisting of 20 sec. 94 ° C denaturation, 30 sec. 65 ° C annealing, 2 min. 72 ° C extension.

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Abstract

L'invention concerne un procédé pour analyser des fragments d'acide nucléique, comprenant les opérations suivantes: a) préparer au moins un mélange de fragments d'acide nucléique comportant au moins un site de reconnaissance pour une endonucléase de restriction coupant en dehors de son site de reconnaissance ; b) faire incuber au moins une partie du mélange des fragments d'acide nucléique obtenu à l'étape (a) avec au moins une endonucléase de restriction dont le site de coupe se trouve en dehors de son site de reconnaissance ; c) identifier une ou plusieurs nucléotides des fragments d'acide nucléique coupés à l'étape (b) et identifier éventuellement d'autres propriétés spécifiques aux fragments des fragments d'acide nucléique coupés à l'étape (b), cette/ces identification(s) se déroulant simultanément pour plusieurs fragments ou pour tous les fragments d'acide nucléique.
EP03742963A 2002-02-27 2003-02-27 Analyse de melanges de fragments d'acide nucleique et l'expression genique Withdrawn EP1492888A2 (fr)

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DE10208333A DE10208333A1 (de) 2002-02-27 2002-02-27 Analyse von Nukleinsäure-Fragmentmischungen
PCT/EP2003/002032 WO2003072819A2 (fr) 2002-02-27 2003-02-27 Analyse de melanges de fragments d'acide nucleique

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US6013445A (en) * 1996-06-06 2000-01-11 Lynx Therapeutics, Inc. Massively parallel signature sequencing by ligation of encoded adaptors
DE19518505A1 (de) * 1995-05-19 1996-11-21 Max Planck Gesellschaft Verfahren zur Genexpressionsanalyse
US6016445A (en) * 1996-04-16 2000-01-18 Cardiotronics Method and apparatus for electrode and transthoracic impedance estimation
US5858671A (en) * 1996-11-01 1999-01-12 The University Of Iowa Research Foundation Iterative and regenerative DNA sequencing method
US6461814B1 (en) * 1997-01-15 2002-10-08 Dominic G. Spinella Method of identifying gene transcription patterns
DE69929542T2 (de) * 1998-10-27 2006-09-14 Affymetrix, Inc., Santa Clara Komplexitätsmanagement und analyse genomischer dna
US6468749B1 (en) * 2000-03-30 2002-10-22 Quark Biotech, Inc. Sequence-dependent gene sorting techniques
US7202022B2 (en) * 2000-06-30 2007-04-10 Syngenta Participations Ag Method for identification, separation and quantitative measurement of nucleic acid fragments

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WO2003072819A2 (fr) 2003-09-04
DE10208333A1 (de) 2003-09-04

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