WO2002029095A1 - Procede d'analyse de mutation - Google Patents

Procede d'analyse de mutation Download PDF

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Publication number
WO2002029095A1
WO2002029095A1 PCT/EP2001/011499 EP0111499W WO0229095A1 WO 2002029095 A1 WO2002029095 A1 WO 2002029095A1 EP 0111499 W EP0111499 W EP 0111499W WO 0229095 A1 WO0229095 A1 WO 0229095A1
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nucleic acid
heterohybrids
acid molecules
group
grappe
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PCT/EP2001/011499
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German (de)
English (en)
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Achim Fischer
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Axaron Bioscience Ag
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Priority claimed from DE10049589A external-priority patent/DE10049589A1/de
Priority claimed from DE10052526A external-priority patent/DE10052526A1/de
Application filed by Axaron Bioscience Ag filed Critical Axaron Bioscience Ag
Priority to AU2002223591A priority Critical patent/AU2002223591A1/en
Publication of WO2002029095A1 publication Critical patent/WO2002029095A1/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
    • 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
    • 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
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase

Definitions

  • the present invention relates to a method for identifying mutations or sequence variations between nucleic acid groups to be compared, the sequence of which may be known or unknown.
  • the nucleic acid groups are provided in a certain way.
  • a composition comprising two such nucleic acid groups is also the subject of the invention.
  • the classic method for recognizing sequence variations in known nucleic acids is sequencing, which today is practically carried out almost exclusively according to the Sanger chain termination principle, but despite recent instrumental advances (e.g. capillary sequencing machines that allow the parallel sequencing of 96 samples) only enables a very low throughput.
  • the single strand cohformation polymorphism detection (SSCP, see Orita et al, Proc. Nati. Acad. Sci. USA 1989, 86 (8): 2766-70), which manages the sequence, is more cost-effective without sequencing - depending on the conformation and thus the mobility of a single nucleic acid strand in a denaturing polyacrylamide gel.
  • the throughput of this method is also limited by the need to provide a separate gel trace for each of the nucleic acid samples to be examined, and the fragments to be examined must not exceed a certain size (100 to 200 base pairs).
  • the method of the oligonucleotide ligation method oligonucleotide ligation assay, OLA; Nickerson et al, Proc. Nati. Acad. Sci. USA 1990, 87 (22): 8923- 1), in which two adjoining oligonucleotides are hybridized to a single-stranded nucleic acid molecule to be examined for a mutation at a specific position.
  • both oligonucleotides can be connected to one another by a ligation. In the case of a mismatch caused by the mutation to be detected, however, no ligation takes place.
  • a variant of the OLA method ligase chain reaction (LCR; W ⁇ edmann et al, PCR Methods Appl. 1994, 3 (4): 51-64), enables signal amplification by amplification, which means detection of mutations in nucleic acids low concentration (such as a single locus within the genome).
  • a disadvantage of OLA is that a separate set of two oligonucleotides must be provided for each position to be checked for a mutation, which can result in high costs.
  • a method was described in which a Sanger sequencing reaction is carried out on a nucleic acid molecule known per se and the products of the primer extension are hybridized with a counter strand which is to be examined for mutation (WO 00/11222).
  • the mismatched nucleotide can be removed by means of a self-correcting polymerase (proofreading polymerase) and replaced by a labeled termination nucleotide, so that detection of the mismatch is possible. Since several steps are necessary to carry out this method in order to check a single pair of two mutually corresponding nucleic acid molecules for sequence differences, this method is also associated with high costs.
  • restriction fragment length polymorphisms restriction fragment length polymorphisms
  • RFLP restriction fragment length polymorphisms
  • a newer principle for identifying sequence variations between nucleic acid molecules originating from two different samples is based on the generation of heterohybrid double strands, one from the first sample and one from each from the second sample, largely complementary strand to be hybridized with each other. Sequence differences between the two strands, for example single base exchanges (single nucleotide polymorphisms, SNPs), lead to mismatches within the double strand, since non-complementary bases are opposed to one another. Such internal mismatches can be chemical (chemical mismatch cleavage, Grompe et al, Proc. Nati. Acad. Sei.
  • the object of the present invention is to provide a method which combines the following advantages:
  • the object of the invention is achieved by a method for analyzing sequence differences between nucleic acid molecules of a first group and a second group, wherein
  • nucleic acid molecules of the first group and the second group flanking sequences which are characteristic of the group and have linkers which are sequence-identical for a large number of nucleic acid molecules of a group; (b) the linkers of the nucleic acid molecules of the first group are to be chosen so that at
  • Formation of hybrids between nucleic acid molecules of the first group and Nucleic acid molecules of the second group, from heterohybrids, starting from nucleic acids of the same group hybridized with one another, from homohybrids, the heterohybridized nucleic acid molecules of both groups, optionally after enzymatic treatment, are a substrate for a double-stranded exonuclease and essentially only the nucleic acid molecules of the first group of a heterohybride be shortened exonucleolytically; with the following steps: (aa) formation of heterohybrids;
  • Another object of the invention is a composition comprising nucleic acid molecules of a first group and a second group with the aforementioned features.
  • the nucleic acid molecules of a first group or a second group are nucleic acid mixtures, preferably nucleic acid mixtures of different origins, in particular nucleic acid mixtures, the sequence information of which can be traced back to genomic DNA, mRNA or cDNA, which was obtained from any different biological samples the term biological sample biological material, which was obtained from one or more individuals.
  • the sequence information of the nucleic acid molecules of the first and the second group can come from tumor biopsies or other biopsies from different patients or patient groups.
  • patient groups can, for example, create groups of patients selected according to certain criteria or certain ethnic groups, such as the North American Amish People or the population of Iceland.
  • nucleic acid molecules of the first and the second group can be derived from the genome of organisms modified by mutagenesis or by spontaneous mutation kind come from.
  • nucleic acid molecules of the first and second groups can be derived from bacterial strains of the same or closely related species that have different properties. Production strains used in biotechnology for the large-scale production of fine chemicals, such as amino acids, are of great economic importance. The determination of the sequence variations between different organism populations can be used to optimize the production processes.
  • the nucleic acid molecules of the first and the second group have flanking sequences, linkers, which are generally characteristic of the group.
  • accompanying means that the characteristic sequences, linkers, occur at both ends of a double-stranded nucleic acid molecule and terminate the nucleic acid molecule towards the ends.
  • the linkers are preferably characteristic of the respective group to which the nucleic acid molecules in question belong. This means that the sequence of the linkers of a single nucleic acid molecule and / or a possible labeling group bound to the linker or another modification, whether the nucleic acid molecule is now double-stranded or single-stranded, can be used to conclude that it belongs to a specific group.
  • the linkers are also sequence-identical for a large number of nucleic acid molecules in a group. This means that in the case of a large number of double-stranded nucleic acid molecules (nucleic acid double strands), the sequences of the linkers match if the sequence of the linker is at the 5 'terminus or at the 3' terminus of the (+) strand or the (-) - Strand of one nucleic acid duplex with the sequence of the linker of the (+) strand or of the (-) strand of the other nucleic acid duplex at the same terminus (at the 5 'terminus or at the 3' terminus) or at the other terminus (on the 3rd 'Term or at the 5' term).
  • a nucleic acid duplex has four linker sequences (at the 5 'terminus and at the 3' terminus of the (+) - and the (-) strand).
  • Two nucleic acid double strands have sequence-identical linkers if the sequence of one of the four linker sequences of the one nucleic acid molecule is identical to one of the four linker sequences of the other nucleic acid molecule.
  • Corresponding considerations also apply to the comparison of the linkers of single-stranded nucleic acid molecules. In this case, the complementary strand is mentally supplemented for the purpose of comparison.
  • not all nucleic acid molecules in a group have to have sequence-identical linkers.
  • the nucleic acid molecules of a group each have a total of no more than 2-4 5 , preferably no more than 2-4 4 , 2-4 3 , 2-4 2 in particular no more than 2-4 3 different sequences characteristic of the group in question.
  • the nucleic acid molecules of a group have only one type of linker or only two types of linker, in the latter case each nucleic acid molecule carrying a linker at one end and a different linker at the opposite end.
  • linkers are generally added to the nucleic acid mixture or nucleic acid mixtures to be investigated by known methods.
  • genomic DNA can be cut with one or more restriction enzymes, and double-stranded oligonucleotides can be added to the fragments obtained using a ligase (Mueller and Wold, Science 1989; 246 (4931): 780-6).
  • the two sequences flanking a fragment can be identical or different from one another.
  • oligonucleotide is to be understood in the broadest sense and encompasses a double-stranded or single-stranded, but preferably double-stranded nucleic acid with 10 to about 100 nucleotide building blocks, which are both naturally occurring and artificially modified or artificially generated nucleotide building blocks amplification by means of PCR is now possible, by using primers which can hybridize with the known flanking sequences.
  • primers which can hybridize with the known flanking sequences.
  • the polymerase chain reaction is to use the promoter sequence of an RNA polymerase, for example introducing T7 RNA polymerase, in one or both of the appended flanking sequences and to perform amplification in the form of an in w 'tro transcription.
  • both ends of a (double) Nuklemklaküls can be selectively provided with various sequences. in the simplest case this is done by cutting the starting nucleic acid molecules with at least two different restriction endonucleases which produce distinguishable ends, for example an overhanging and a smooth end or a 3 'overhanging end and 5' overhanging end. Then specific and different oligonucleotides are added for the respective end, each forming different linkers. Subsequent amplification with primers which can hybridize to different linkers, using the known PCR suppression effect (Luk'ianov et al, Bioorg. Khim.
  • Such representations can be generated, for example, by cutting genomic DNA with less frequently cutting restriction endonucleases with a recognition sequence which comprises 6, 8 or more bases, followed by a size selection, for example all those fragments whose length does not exceed 1 kb.
  • Another way to generate representations is to use a less frequently cutting restriction endonuclease in combination with a more often cutting restriction endonuclease, followed by obtaining fragments with different ends.
  • representations are produced in such a way that a set of non-overlapping fragment populations is generated, which in its entirety comprises all or at least largely all of the sequence regions contained in the starting nucleic acids.
  • fragment the starting nucleic acids With the aid of a restriction endonuclease which cuts sufficiently frequently or a mixture of restriction endonucleases which cuts sufficiently frequently in order to generate fragments in the size range of a few hundred base pairs.
  • This fragment population now initially comprises the entire complexity of the starting nucleic acids.
  • double-stranded oligonucleotides are added by ligation, the oligonucleotides inserting the sequence of the later linkers, followed by PCR amplification with primers that hybridize with the sequences of the linkers can.
  • restriction endonucleases that cut within their recognition site can also be used, the use of type IIS restriction endonucleases is particularly advantageous. These restriction endonucleases cut outside their recognition site and create an overhang of defined length and position relative to the recognition site.
  • double-stranded oligonucleotides are added on both sides to the fragments produced.
  • a set of oligonucleotides is provided which contains all possible overhangs of the type specified by the respective restriction endonuclease (i.e. 5 'overhangs or 3' overhangs of a defined length), the sequence of the oligonucleotides, i.e.
  • the later linker sequences being so different from one another distinguishes that they do not form heterohybrids with each other.
  • the Li gation takes place under conditions which suppress a linkage of overhangs which are not completely complementary to one another (Shaw-Smith et al, Biotechniques 2000, 28 (5): 958-64).
  • a further possibility for generating representations consists in amplifying left-flanked nucleic acid fragments by means of primers, so-called selective primers, which are extended at their 3 'end beyond the sequence specified by the linkers, and which allow amplification only of those nucleic acid fragments with which they in particular perfectly base pair can enter at their 3 'end formed by the selective bases.
  • representations of genomic DNA can be generated by generating chromosome-specific nucleic acids in a known manner (e.g. with Fluss-Karyotypie, Harris et al, Hum. Genet. 1985; 70 (l): 59-65).
  • the nucleic acids obtained in this way are provided with linkers, for example as described above by restriction with type IIS restriction endonucleases and subsequent ligation with overhang-specific oligonucleotides which contribute the later linker sequences.
  • the nucleic acid molecules of the first and second groups are thus generally obtained in the same way, but preferably differ by the sequence of their linkers, which according to the invention is characteristic of the group in question, and by the sequence variations, the detection of which is the subject of the method according to the invention ,
  • the linkers of the nucleic acid molecules of both groups are generally to be chosen such that when hybrids are formed between nucleic acid molecules of the first group and nucleic acid molecules of the second group, starting from heterohybrids, starting from hybridized nucleic acids of the same group, homohybrids, the heterohybridized linkers of the nucleic acid molecules of both Groups are sequence complementary over at least one sub-area.
  • hybrid is understood to mean a dimer of nucleic acids with the formation of base pairings.
  • Hybrids between nucleic acid molecules of the first group and nucleic acid molecules of the second group are generally formed by melting the double strands of nucleic acids of the same group hybridized with one another (from homohybrids) and subsequent cooling, whereby not only nucleic acid molecules and recombine the same group, but also heterohybrids are formed.
  • this recombination process that is, whether the kinetic or thermodynamic control of the reaction predominates, the proportion of hybrids that have mismatches varies.
  • Optimal conditions consist in a maximum yield of heterohybrids with a minimal proportion of by-products, i.e. hybrids from nucleic acid molecules that are not homologous.
  • homology means a close relationship between the nucleic acids in question, so that the sequence-complementary region of the two nucleic acid molecules is considerably more extensive than the region occupied by the sequence variations.
  • a nucleic acid molecule (or a region thereof) is sequence complementary to another nucleic acid molecule (or a region thereof) if it can form base pairings with the other molecule (or a region thereof) without mismatches, that is to say perfect base pairings.
  • the linkers of both groups which are opposed to each other in the heterohybrid, can generally form base pairing at least over a sub-area.
  • the linkers which have a homohybrid at both ends need not necessarily be attributed to the same oligonucleotides.
  • the partial range of the sequence complement generally comprises at least 4 bases, preferably at least 10 bases, in particular at least 20 bases.
  • linkers of the nucleic acid molecules of both groups are also to be chosen such that the heterohybrids, if appropriate after enzymatic treatment, represent a substrate for a double-stranded exonuclease and essentially only the nucleic acid molecules of the first group of a heterohybride are exonucleolytically shortened.
  • a double-strand-specific exonuclease is an enzyme which degrades a nucleic acid molecule or both nucleic acid molecules of a nucleic acid double-strand in the region of the double-strand which is double-stranded from a terminus of the respective nucleic acid molecule.
  • Exonuclease III shortens one or both nucleic acid molecule (s) of a double strand from the 3 'terminus.
  • the 3 'terminus of a nucleic acid molecule is only shortened if the 3' Terminus together with the other nucleic acid molecule forms a smooth end or a 3'- recessed end (i.e. a 5 'overhang).
  • the heterohybrids do not have to be a substrate for the double-stranded exonuclease in the state in which they are formed. It is possible to incubate the heterohybrids with another enzyme such as a restriction endonuclease (enzymatic treatment) before they are reacted with the double-strand-specific exonuclease. Then, as a rule, the heterohybrids only become a substrate for the double-strand-specific exonuclease as a result of this enzymatic treatment.
  • another enzyme such as a restriction endonuclease (enzymatic treatment)
  • the heterohybrids can be incubated with a restriction endonuclease or with restriction endonucleases which cut in the region of the linkers, that is to say in the regions of the nucleic acid molecules.
  • the linkers can be selected so that there is a restriction site for the restriction enzyme or the restriction enzymes with which the enzymatic treatment is carried out in the case of homohybrids but not in the case of heterohybrids or vice versa.
  • heterohybrids are a substrate for a double-stranded exonuclease and that essentially only the nucleic acid molecules of the first group of a heterohybride (that is to say are the constituents of a heterohybrid) and not the nucleic acid molecules of the second group of a heterohybride are exonucleolytically shortened.
  • homohybrids which are present in addition to the heterohybrids, should not form a substrate for the double-stranded exonuclease.
  • nucleic acid molecules of the first group of a heterohybrid are shortened exonucleolytically. As far as there is an exonucleolytic shortening of the nucleic acid molecules of the second group, this reaction is only a negligible side reaction.
  • a composition comprising nucleic acid molecules of a first group and a second group as described above is an object of the invention.
  • Another object is a method for analyzing sequence differences between nucleic acid molecules of a first group and a second group, as described above, with the following steps: (aa) formation of heterohybrids;
  • the method according to the invention thus initially comprises the formation of heterohybrids, as already described. If the homohybrids represent a substrate for the double-stranded exonuclease in addition to the heterohybrids, which is not excluded according to the invention, then these homohybrids can be separated. This can be done by a variety of measures. A suitable measure would be the separation of the homohybrids from the heterohybrids by the different electrophoretic mobility of homo- and heterohybrids.
  • an enzymatic treatment can take place, for example a restriction cut in the region of the linkers, which takes place either only in homohybrids or only in heterohybrids, and the separation of homohybrids from selective groups which can be immobilized the heterohybrids.
  • Such a separation is not necessary, for example, if the heterohybrids are converted into a substrate for the double-strand-specific exonuclease by the action of the restriction endonuclease ⁇ ), while the homohybrids do not become a substrate for the double-strand-specific endonuclease due to the restriction cut which is not carried out.
  • the linkers carry nucleotides which exclude degradation by a double-stranded exonuclease and which are separated from the heterohybrids as a result of a restriction cut, but not from the homohybrids.
  • nucleic acid molecules of the first group of heterohybrids with the aid of the double-strand-specific exonuclease, preferably from the 3 'terminus of the nucleic acid molecule to be degraded in the direction of the 5' terminus.
  • nucleic acid molecules of the first group are essentially exclusively exonucleolytically shortened, while the nucleic acid molecules of the second group remain unaffected.
  • exonucleolytic shortening of nucleic acid molecules of the second group of heterohybrids if it takes place, remains a negligible side reaction.
  • the exonucleolytic truncation of nucleic acid molecules in the first group preferably takes place in such a way that a large number of heterohybrids are formed in which the nucleic acid molecule in question in the first group has been truncated to different extents, and the truncation products are present in as many copies as possible. Accordingly, the collection of the truncated variants of a given nucleic acid molecule with a length of 100 bp, for example, ideally contained all truncated molecules with a length of 1 bp to 99 bp at their 3 'end, the linker being ignored here.
  • the reaction conditions in particular the reaction time, temperature and amount of the exonuclease used, are chosen such that complete strand degradation does not take place.
  • a very important factor is the processivity of the exonuclease. If the processivity is very high, complete strand degradation is preferred. If the processivity is very low, the shortening products show essentially the same degree of shortening. Using genetic engineering methods, the processivity of the exonuclease must be adjusted so that it lies between these extremes.
  • processivity encompasses the number of those nucleotide building blocks that are broken down on a statistical average before the enzyme dissociates from the double-stranded nucleic acid.
  • a particularly uniform distribution of the shortening products can also be achieved in the nucleic acid molecule to be degraded by the statistical incorporation of nucleotides which are resistant to degradation by double-strand-specific exonucleases. These can be, for example, ⁇ S thionucleotides (Schreiber et al, Nucleic Acid Res. 1985; 13 (21): 7663-72).
  • the double-strand-specific exonuclease can be used in excess, so that complete exonucleolytic degradation takes place up to the respective degradation-resistant nucleotide.
  • heterohybrids can also be obtained by generating the shortened nucleic acid molecules of the first group as described in WO 00/11222 by a Sanger sequencing reaction.
  • shortened nucleic acid molecules of the first group with unabridged nucleic acid molecules of the second group is not preferred in the context of this invention, since it differs Distinguish long nucleic acid molecules in their hybridization kinetics and also very short nucleic acid molecules tend to non-specific hybridization (“cross hybridization”), which could impair the efficiency of the method.
  • heterohybrids which have a single- or polybasic mismatch at the 3 'terminus of the nuclear acid molecule of the first group, the mismatched heterohybrids, from the perfectly paired heterohybrids.
  • the distinction is usually based on the known ability of certain nucleic acid polymerases to recognize mismatches at the 3 'terminus of a nucleic acid molecule and to exclude such molecules from strand extension.
  • a DNA or RNA polymerase can be used as the polymerase, which is able to extend set back 3 'terms, but not to extend mismatched terms, but not polymerases which can reduce terminal mismatches with exonuclease activity (proofreading polymerases).
  • suitable polymerases are, for example, genetically modified T7 DNA polymerases ( ⁇ 28T7 DNA polymerase), Taq polymerase or reverse transcriptase.
  • native T7 DNA polymerase or Pfu polymerase are not suitable.
  • proofreading polymerases can of course also be used. In a preferred embodiment of the method according to the invention, the distinction is made
  • the perfectly paired heterohybrids are filled in by a DNA polymerase, which extends the shortened nucleic acid molecule in the 5 '-3' direction, i.e. fills in until a continuous double strand with a mostly smooth end is obtained.
  • the filled heterohybrids are separated from the unfilled, mismatched heterohybrids. This can be done by using nucleotide monomers which carry immobilizable groups in the replenishment reaction in the previous step, so that the products of the replenishment reaction themselves can be immobilized and thus differentiated from the hetero-hybrids that have not been replenished, the mismatched heterohybrids can be.
  • the non-filled heterohybrids are identified by known methods, for example by hybridization with an arrangement of single-stranded nucleic acids on a surface. This can be followed by known sequencing methods, such as the Sanger sequencing method (strand-lengthening) or Brenner (strand-shortening, US Pat. No. 5,846,719).
  • nucleotides of a first type have an immobilizable group that do not have the nucleotides of the second type, or the nucleotides of the first type do not have an immobilizable group that have the nucleotides of the second type;
  • mismatched base (s) of the mismatched heterohybrids followed by the replenishment of the perfectly paired heterohybrids, can of course be replaced by replenishment of mismatched heterohybrids if the polymerization conditions set for replenishment are sufficiently permissive, i.e. the most complete possible extension of mismatched heterohybrids by allow the chosen polymerase.
  • the Klenow fragment of DNA polymerase I from E. coli is able to efficiently replenish mismatched heterohybrids at high nucleotide and magnesium concentrations (for example 200 ⁇ M dNTPs / 2 mM MgCl 2 ).
  • the immobilizable group is preferably biotin, which enables immobilization via streptavidin.
  • Other examples of immobilizable groups are sugars that can be recognized by lectins, or molecular groups that allow immobilization by means of antibodies binding the molecular groups. Examples of the latter would be digoxigenin or dinitrophenol derivatives.
  • Such immobilizable groups can of course also be used as so-called caged compounds (Methods Enzymol. 1998; 291: 415-30), in which the immobilizable group is only released as a result of a photo reaction.
  • the perfectly paired heterohybrids are filled in while maintaining a continuous double strand as described above. This means that only the mismatched heterohybrids have a non-continuous double strand.
  • This enables hybridization with an immobilized or immobilizable oligonucleotide and the subsequent immobilization of the hybrid of heterohybrid and oligonucleotide to a solid phase, as a result of which the mismatched heterohybrids can be separated from the originally perfectly paired heterohybrids.
  • the isolated, originally mismatched heterohybrids are identified by known methods such as the sequencing methods described above.
  • the 3 'terminus of the perfectly paired heterohybrids is extended by a nucleotide leading to chain termination.
  • a preferred nucleotide leading to chain termination is a dideoxynucleotide such as ddATP, ddCTP, ddTTP and ddGTP.
  • the nucleotides which are not incorporated and which lead to chain termination are optionally removed (optional).
  • the mismatched base (s) of the mismatched heterohybrids are preferably removed with the participation of a self-correcting DNA polymerase (proofreading polymerase).
  • the perfectly paired heterohybrids formed in the previous step are then filled in to form a continuous double strand, no nucleotides leading to chain termination being used.
  • the heterohybrids, which are in the form of a double strand are then isolated, for example on the basis of their electrophoretic mobility or by cloning. If the originally mismatched heterohybrids are isolated by cloning, the heterohybrids present in the form of a continuous double strand become at both ends with a restriction enzyme, preferably in the region of the linker, cut and ligated with a correspondingly prepared vector and then cloned. Linker or restriction endonucleases are to be selected in such a way that double strands which are not continuous cannot be cut on both sides by the restriction endonucleases.
  • the isolated heterohybrids are identified by known measures.
  • the nucleotide leading to chain termination carries an immobilizable grappe and the heterohybrids present in the form of a double strand are isolated by immobilization and separation of the heterohybrids extended by an immobilizable group.
  • the immobilizable group can be, for example, a biotin group, which can be immobilized by binding to streptavidin.
  • the homohybrids of step (bb) are cut in the region of the linkers, the restriction site being present only in the case of homohybrids, but not in the case of heterohybrids.
  • the reverse is the case.
  • the restriction interface is only available for heterohybrids, but not for homohybrids.
  • linkers have nucleotides which are resistant to exonucleolytic degradation and which are separated in heterohybrids but not in homohybrids as a result of the restriction, then only the heterohybrids are a substrate for the double-strand-specific exonuclease, so that the homohybrids are separated in step (bb ) can be omitted.
  • the linker of the nucleic acid molecule of the second group should carry a nucleotide resistant to exonucleolytic degradation in the vicinity of the 3 'terminus, that is to say in the region of the linker, in order to ensure that essentially only the nucleic acid molecules of the first group of a heterohybrid shorten exonucleolytically ): become.
  • the linkers of the nucleic acid molecules of the first Grappe are chosen so that when heterohybrids are formed, the linkers of the nucleic acid molecules of both groups form a 5 'overhang and a 3' overhang and both overhangs each by the nucleic acid molecules of the second Group are formed.
  • the easiest way to achieve this is that the linkers of the nucleic acid molecules of the second group of a heterohybrid are longer than the linkers of the nucleic acid molecules of the first group of the same heterohybride.
  • the homohybrids have 3 'overhangs on both sides, so that a separation can be dispensed with.
  • the mismatched heterohybrids are distinguished from the perfectly paired heterohybrids by amplification.
  • the filled-in, perfectly paired heterohybrids can be cut in the region of one of the two linkers by means of one or more specifically double-stranded DNA-cutting restriction endonuclease (s), so that the complete linker concerned or a partial region of this linker is removed. Since the corresponding area is single-stranded in the mismatched, unfilled heterohybrids, it is not separated from them in the restriction step.
  • linker strands in the heterohybrid containing non-functional, “masked” restriction sites, that is to say interfaces which are only present in a homohybrid but not in a heterohybrid linker in a fractional state.
  • the reaction sequence “exonucleolytic shortening - Replenishment of the perfectly paired heterohybrids ", the linker strand, which has become single-stranded after shortening, is supplemented by its reverse complement to the double strand, which now contains an" unmasked ", ie functional interface.
  • the linker at the opposite end of the same heterohybride remains non-functional, so that only shortened and then replenished heterohybrids can be cut at one end in the linker (in the "unmasked” interface): NNN GGATCC GGTACCNNN NNN CCATGG CCTAGGNNN
  • the mismatched heterohybrids are also filled (see Fig. 15), the amplification product contains a mixture of both sequence variants of the corresponding nucleic acid molecule. If, on the other hand, the mismatched heterohybrids are not filled in, only the strand which has remained unabridged and which comes from nucleic acid molecules of the second group is amplified.
  • a procedure analogous to the result for distinguishing mismatched from perfectly paired heterohybrids is, after successful exonucleolytic shortening and extension of the perfectly paired heterohybrids, by a nucleotide leading to chain termination and subsequent filling of the mismatched heterohybrids under permissive conditions or using a proofreading polymerase complete double strand to remove single-stranded overhangs. Since the perfectly paired heterohybrids, which are extended by a kettan-terminating nucleotide, have a single-stranded overhang encompassing the region of one of the linkers, can also be removed in this way, primer binding sites not to amplify desired nucleic acid molecules (see Fig. 14)
  • linkers as binding sites for amplification primers could also only be carried out after shortening and differentiation of mismatched heterohybrids from perfectly paired heterohybrids.
  • nucleic acid molecules isolated from the respective individuals of a patient group become nucleic acid molecules of the first Grappe first Origin combined and the nucleic acid molecules isolated from the respective individuals of a second patient group are combined to form nucleic acid molecules of the first Grappe of second origin.
  • nucleic acid molecules of a further origin are provided as a "reference", which are obtained, for example, from an individual who does not belong to either of the above two groups or from a cell culture.
  • patient group is understood not only as an individual group of human patients, but also as a group of individual animals, plants, cells or tissues.
  • the method according to the invention is then carried out in parallel batches, the nucleic acids obtained from one patient group (first origin) in one of the batches and the nucleic acids obtained from the other patient group (second batch) in the other batch ) are used as nucleic acid molecules of the first group.
  • the reference nucleic acid molecules serve as nucleic acids of the second group.
  • the method according to the invention is then used to isolate nucleic acid molecules which, in the respective batch, have sequence differences from the reference nucleic acid molecules provided.
  • the procedure is preferably such that the isolated nucleotides Acid molecules of the two patient groups to be compared (first and second origin) obtained markings distinguishable from each other in both approaches.
  • the unfilled heterohybrids obtained in step (6b) could be labeled with the fluorescent dye Cy3 in the case of the first patient group and with the fluorescent dye Cy5 in the case of the second patient group.
  • An arrangement of reference nucleic acid molecules would then be provided, for example in the form of an array on a solid surface, which can take place in a conventional manner by depositing nucleic acid clones in a regular arrangement or by surface amplification following a random arrangement, or in the form of particles loaded with identical nucleic acid molecules.
  • the arrangement should have the properties already described above that identical and hybridization-capable nucleic acid molecules are localized at one location. Location can mean both an area on a mostly planar surface and the surface of a particle.
  • the differently labeled nucleic acid molecules described above which each represent sequence differences between nucleic acids of the first patient group (first origin) and the reference or between nucleic acids of the second patient group (second origin) and the reference, would be hybridized with said arrangement. After evaluation, four different situations can be distinguished: (1) a fragment from the first patient group is identical to the sequence both with the corresponding fragment from the second patient group and with the respective reference fragment.
  • a fragment from the second patient group has a sequence difference compared to the respective reference fragment, while the corresponding fragment from the first patient group is sequence-identical with the respective reference fragment. Therefore, only probe molecules are obtained that carry a label that identifies a sequence difference between the reference and the second patient group, in the example above, that is, a Cy5 label. After hybridization, only a signal originating from this (Cy5) label is obtained from the locations of the arrangement belonging to this fragment.
  • the fragments from the first and second patient groups have a sequence difference compared to the respective reference fragment.
  • both probe molecules are obtained which have a marking which distinguishes a sequence difference between the reference and the first group of patients, and probe molecules which have a marking which distinguishes a sequence difference between the reference and the second group of patients.
  • a signal originating from both labels is therefore obtained from the locations of the arrangement belonging to this fragment.
  • nucleic acid molecules located at those locations are therefore identified which are distinguished by a hybridization signal obtained only or predominantly from one of the two probe preparations.
  • sequence differences exist within a patient group. This will usually be the case, for example, if nucleic acid molecules are obtained from different, non-clonal individuals and interact with one another. who are united. For example, a certain allele of a given gene within a patient group can occupy all relative frequencies between 0% and 100%. If the relative frequency of this allele differs between the two individual patient groups, probe molecules derived from the corresponding fragment and representing the first or the second patient group are obtained in different amounts, so the hybridization leads to mixed signals at the corresponding locations of the arrangement.
  • the method according to the invention therefore not only allows the identification of sequence differences that occur in a first group of patients and does not occur in a second patient group, but also the identification of sequence differences that occur in different patient groups at different frequencies.
  • the shortened heterohybrids can also be produced using an exonuclease with 5 ' ⁇ 3' exonuclease activity.
  • the exonucleolytic degradation of only one strand of nucleic acid can preferably be achieved using the exonuclease with 5 '->3'-exonuclease activity in such a way that one of the linkers has incorporated ⁇ -thio-nucleotides which have an exonucleolytic degradation of the strand prevent who has said linker sequence at its 5 'end.
  • This further preferred embodiment of the method according to the invention consists in that the nucleic acid molecules of the first group are shortened by means of an exonuclease working in the 5 ' ⁇ 3' direction, for example T7 exonuclease or lambda exonuclease, followed by an extension of a linker primer by means of a polymerase without strand displacement activity and a ligation of the extension products to the shortening products to distinguish perfectly paired heterohybrids from heterohybrids, which are characterized by a terminal mismatch.
  • an exonuclease working in the 5 ' ⁇ 3' direction for example T7 exonuclease or lambda exonuclease
  • an extension of a linker primer by means of a polymerase without strand displacement activity and a ligation of the extension products to the shortening products to distinguish perfectly paired heterohybrids from heterohybrids, which are characterized by a terminal mismatch.
  • Such a modification would include in step (dd) a distinction between those heterohybrids that were at the 5 'terminus of the nucleic acid molecule of the first grappe have a single- or multi-base mismatch, the mismatched heterohybrids, of the perfectly paired heterohybrids.
  • ligases in the case of interruptions of one of the two sugar-phosphate chains, single-strand breaks, present in a nucleic acid double strand, to differentiate between perfectly paired 5 'terms and mismatched 5' terms by perfectly paired 5 'terms with the neighboring 3' -Termini are connected by ligation, while mismatched 5 '-terminini are not connected to the neighboring 3'-termini.
  • the subsequent separation of perfectly paired ligated heterohybrids from unmatched mismatched heterohybrids could be done, for example, by one of the following measures:
  • FIG. 2 shows the production of heterohybrids with a shortened strand
  • FIG. 3 shows the isolation of a fragment from a mixture of nucleic acid molecules which has a sequence variation between the nucleic acid molecules of the first and the second group
  • FIG. 4 shows an alternative isolation of a fragment from a mixture from nucleic acid molecules which has a sequence variation between the nucleic acid molecules of the first and the second group
  • FIG. 5 shows a further alternative isolation of a fragment from a mixture of nucleic acid molecules which has a sequence variation between the nucleic acid molecules of the first and the second group
  • FIG. 6 shows a further alternative isolation of a fragment from a mixture of nucleic acid molecules which has a sequence variation between the nucleic acid molecules of the first and the second group
  • FIG. 7 shows a further alternative isolation of a fragment from a mixture of nucleic acid molecules which has a sequence variation between 8
  • FIG. 9 shows a further alternative isolation of a fragment from a Mixture of nucleic acid molecules which has a sequence variation between the nucleic acid molecules of the first and second grappa
  • FIG. 10 shows the selective restriction of homohybrids in the region of the linkers
  • FIG. 11 shows the selective restriction of homohybrids in the region of the link he followed by isolation of a heterohybride carrying a sequence variation and preparation of a hybridization probe
  • FIG. 12 shows the use of the method according to the invention for the identification of sequence variations between nucleic acids originating from two different samples using reference nucleic acids
  • FIG. 12 includes Fig. 12a,
  • FIG. 14 shows an alternative isolation of a fragment from a mixture of nucleic acid molecules which has a sequence variation between the nucleic acid molecules of the first and the second grappa (FIG. 14 includes FIGS. 14a and FIG 14b, which form a coherent Fig. 14),
  • FIG. 15 shows a further alternative isolation of a fragment from a mixture of nucleic acid molecules which has a sequence variation between the nucleic acid molecules of the first and the second grappa
  • FIG. 15 comprises FIGS. 15a and 15b, which form a coherent FIG. 15
  • 16 shows a further alternative isolation of a fragment from a mixture of nucleic acid molecules which has a sequence variation between the nucleic acid molecules of the first and the second group.
  • Fig. 1 shows the production of heterohybrids, which enable a selective shortening of only one strand. It shows in detail
  • Fig. 2 shows the production of heterohybrids with a shortened strand, shows in the eggshell
  • 3 shows the isolation of a fragment from a mixture of nucleic acid molecules which has a sequence variation between the nucleic acid molecules of the first and the second group.
  • the mixture here consists of two fragments, of which the shorter one between the first and second origin is sequence-identical, while the longer one has a sequence variation between the first and second origin. 1 shows the extraction of heterohybrids
  • Shortening product represents, whose last nucleotide of the double-stranded region represents the sequence variation between nucleic acid molecules of both grappas (point). This shortening product is therefore characterized by a mismatch at the end of the double-stranded area,
  • Biotin grapples are represented by "B", 4 separation of biotin-containing heterohybrids from unextended, non-biotin-containing heterohybrids, which represent fragments containing sequence variations.
  • FIG. 5 shows a further alternative isolation of a fragment from a mixture of nucleic acid molecules which has a sequence variation between the nucleic acid molecules of the first and the second grappa.
  • 6 shows a further alternative isolation of a fragment from a mixture of nucleic acid molecules which has a sequence variation between the nucleic acid molecules of the first and the second group. 1 shows the extraction of heterohybrids
  • FIG. 7 shows a further alternative isolation of a fragment from a mixture of nucleic acid molecules which has a sequence variation between the nucleic acid molecules of the first and the second grappa.
  • Linkers were attached to nucleic acid fragments (gray) generated using the restriction endonuclease Hi ⁇ Pll (recognition sequence GCGC).
  • Linkers of the fragments of the first group (open rectangles) contain a recognition site for the restriction endonuclease H ⁇ ell (recognition sequence AGCGCT), linkers of the fragments of the second group (hatched) an alternative recognition site for the same restriction endonuclease GGCGCC.
  • Sequence variations between nucleic acids from two different samples using reference nucleic acids A certain nucleic acid fragment flanked by linker molecules is shown, which consists of the first sample originating from short hatched linkers, from the second sample originating from short dotted linkers and from the reference sample originating from long rectangles represented as open rectangles.
  • the position of a sequence variation (SNP) between the fragment originating from the first sample and the fragments originating from the second sample or the reference sample is identified by a dot.
  • the fragments from the reference sample used for the described implementation with fragments from the first sample are provided with a marker group, which is symbolized by an asterisk.
  • the fragments used for the described implementation with fragments from the second sample and originating from the reference sample are provided with a marking group which can be distinguished from the first marking range, which is symbolized by "#"
  • 10 means: no signal, since there is no sequence variation between the first and second sample and reference sample; 11: signal indicates sequence variation between first sample and reference sample; 12: signal indicates sequence variation between second sample and reference sample; 13; Signal indicates sequence variation, both between the first sample and reference sample and between the second sample and reference sample.
  • Biotinylated linkers are attached to nucleic acid fragments generated by means of the restriction endonuclease Dpril (recognition sequence GATC).
  • the linkers contain masked recognition sequences for the restriction endonuclease Baml ⁇ l (recognition sequence GGATCC).
  • the linkers attached to the nucleic acid molecules of the second group have a thionucleotide in the vicinity of the recognition sequence (denoted by “S”).
  • 1. shows the denaturation of the nucleic acid fragments flanked by linkers of both grappas, followed by a re-hybridization of strands that are partially complementary to one another to homo and hetero hybrids,
  • 15 shows a further alternative isolation of a fragment from a mixture of nucleic acid molecules, which has a sequence variation between the nucleic acid molecules of the first and the second group. 1 shows the extraction of heterohybrids,
  • a proofreading polymerase could also be used, which first removes the terminal mismatch), 6 the amplification of the double strands flanked in (5) to both sides by linkers by means of amplification primers, which can bind to the linker regions of the nucleic acid molecules, which essentially match those in the in (3) filled heterohybrids in (4) distant areas.
  • 16 shows a further alternative isolation of a fragment from a mixture of nucleic acid molecules which has a sequence variation between the nucleic acid molecules of the first and the second group.
  • 1 shows the production of heterohybrids
  • 2 shows the selective exonucleolytic shortening of the nucleic acid molecules of the first group in the heterohybrids from (1) in the 5 ' ⁇ 3' direction
  • FIG. 3 shows the hybridization of an oligonucleotide primer to the single-stranded linker region, followed by an extension by means of a polymerase having no beach displacement activity, wherein nucleotide building blocks containing biotin are incorporated,
  • step 5 the further extension of the prime extension products from step (3) which remained unligated in step (4) by means of a polymerase with strand displacement activity, followed by immobilization by binding to a streptavidin-coated solid phase, followed by the removal of the nucleic acid molecules of the second group hybridized to the extension products and a subsequent amplification of the immobilized nucleic acid strands by means of amplification primers which hybridize specifically with the linker regions.
  • the invention is described below by examples:
  • oligonucleotides DL20 (5'-GCA TCA CAA GAA TCG ACG CT-3 '), LD24 (5'-phosphate-GAT CAG CGT CGA TTC TTG TGA TGC-3'), ML25BGL (5'-TCA CAT) were supplied lyophilized GCT AAG TGA CGT AGA TCT T-3 '), LM33BGL (5'-Phosphate-GAT CAA GAT CTA CGT CAC TTA GCA TGT GAC AAT-3'), ML33BAM (5'-GCT CAT TGT CAC ATG CTA AGT GAC GTG GAT CCT-3 ') and LM41BAM (5'-phosphate-GAT CAG GAT CCA CGT CAC TTA GCA TGT GAC AAT GAG CAT CG-3') to a final concentration of 100 pmol / ⁇ l in water.
  • the finished linkers DL2024 (from DL20 and LD24), ML2533BGL (from ML25BGL and LM33BGL) and ML3341BAM (from ML33BAM and LM41BAM) were frozen at -20 ° C until use stored.
  • the samples were extracted with phenol, then with chloroform and precipitated with ethanol. It was taken up in 10 ⁇ l of a ligation mixture containing 0.6 ⁇ l lOx ligation buffer (Röche), 1 ⁇ l 10 mM ATP (Röche), 4 ⁇ l 0.5 ⁇ g / ⁇ l linker DL2024, 3.9 ⁇ l H 2 O and 0, 5 ul T4 DNA ligase (Röche). The ligation took place at 16 ° C. overnight. Unligated and self-ligated linkers were separated by centrifugation through ChromaSpin 100 columns (Clontech Inc., Palo Alto, CA, USA).
  • One-tenth of the left-flanked fragments of genomic DNA obtained in Example 2 were used as templates in a 100 ⁇ l amplification mixture containing 10 ⁇ l 10 ⁇ AmpHTaq buffer (PE Applied Biosystems, Foster City, CA, USA), 2 mM MgCl 2 , 0, 4 mM oligonucleotide DL20, 200 ⁇ M dNTPs, and 2.5 U AmpHTaq DNA polymerase (PE Applied Biosystems).
  • One of the preparations (preparation 1) was 10% of each nucleotide replaced by its respective ⁇ -thioanalog (Amersham-Pharmacia GmbH, Freiburg).
  • An amplification was carried out with the following temperature program: 1 min 95 ° C, then 25 cycles consisting of 30 sec 95 ° C, 30 sec 60 ° C, 1 min 72 ° C.
  • the amplification was checked using agarose gel electrophoresis, then the reactions were purified using QiaQuick columns (Qiagen GmbH, Hilden).
  • the eluted amplification products were taken up in 50 ⁇ l NEBuffer Dp ⁇ ll, mixed with 5 U Dpn ⁇ l and incubated for 1.5 h at 37 ° C.
  • the cut linkers were removed using ChromaSpin 100 columns and the eluted DNA fragments were precipitated with ethanol.
  • the pellets were taken up in 10 ⁇ l ligation batches, containing 0.6 ⁇ l 10 ⁇ ligation buffer, 1 ⁇ l 10 mM ATP, 4 ⁇ l 0.5 ⁇ g / ⁇ l linker, 3.9 ⁇ l H 2 O and 0.5 ⁇ l T4 DNA - ligase.
  • Linker ML2533BGL was used for the fragments of preparation 1 and linker ML3341BAM for preparation 2. The ligation took place at 16 ° C. overnight. Unligated and self-ligated linkers were separated by centrifugation through ChromaSpin 200 columns (Clontech).
  • the amplification products obtained were checked by means of agarose gel electrophoresis and used for cloning in a T / A vector (pCR2.1 TOPO, Invitrogen, Groningen, NL).
  • the clones obtained were sequenced and used to design PCR primers for verifying the identified regions, which were identified as having sequence variations between genomic DNA of preparation 1 and preparation 2.

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Abstract

L'invention concerne un procédé permettant d'analyser des séquences différentielles entre des molécules d'acide nucléiques d'une premier et d'un second groupe, qui comprend les étapes suivantes : (aa) former des hétérohybrides ; (bb) éventuellement séparer des homohybrides ; (cc) réduire par voie exonucléotique des molécules d'acide nucléique du premier groupe d'un hétérohybride à l'aide de l'exonucléase à brin double spécifique ; (dd) différencier les hétérohybrides qui présentent à l'extrémité 3' de la molécule d'acide nucléique du premier groupe, un appariement défectueux à une ou plusieurs bases, les hétérohybrides mal appariés, des hétérohybrides parfaitement appariés.
PCT/EP2001/011499 2000-10-06 2001-10-05 Procede d'analyse de mutation WO2002029095A1 (fr)

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Citations (4)

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Publication number Priority date Publication date Assignee Title
WO1998042871A1 (fr) * 1997-03-24 1998-10-01 Boehringer Mannheim Corporation Procede d'hybridation soustractive et analyse differentielle
US5879886A (en) * 1993-09-10 1999-03-09 Institut Pasteur Method for detecting molecules containing nucleotide mismatches and the location of these mismatches, and application to the detection of base substitutions or deletions in nucleotide sequences
WO2000046402A1 (fr) * 1999-02-05 2000-08-10 Amersham Pharmacia Biotech Uk Limited Technique d'analyse genomique
DE19911130A1 (de) * 1999-03-12 2000-09-21 Hager Joerg Verfahren zur Identifikation chromosomaler Regionen und Gene

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Publication number Priority date Publication date Assignee Title
US5879886A (en) * 1993-09-10 1999-03-09 Institut Pasteur Method for detecting molecules containing nucleotide mismatches and the location of these mismatches, and application to the detection of base substitutions or deletions in nucleotide sequences
WO1998042871A1 (fr) * 1997-03-24 1998-10-01 Boehringer Mannheim Corporation Procede d'hybridation soustractive et analyse differentielle
WO2000046402A1 (fr) * 1999-02-05 2000-08-10 Amersham Pharmacia Biotech Uk Limited Technique d'analyse genomique
DE19911130A1 (de) * 1999-03-12 2000-09-21 Hager Joerg Verfahren zur Identifikation chromosomaler Regionen und Gene

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YANG M ET AL: "CLONING DIFFERENTIALLY EXPRESSED GENES BY LINKER CAPTURE SUBTRACTION", ANALYTICAL BIOCHEMISTRY, ACADEMIC PRESS, SAN DIEGO, CA, US, vol. 237, no. 1, 15 May 1996 (1996-05-15), pages 109 - 114, XP000587696, ISSN: 0003-2697 *
ZENG J ET AL: "DIFFERENTIAL CDNA CLONING BY ENZYMATIC DEGRADING SUBTRACTION (EDS)", NUCLEIC ACIDS RESEARCH, OXFORD UNIVERSITY PRESS, SURREY, GB, vol. 22, no. 21, 1994, pages 4381 - 4385, XP002039836, ISSN: 0305-1048 *

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