EP1360333A2 - Vergleichende auswertung der nukleinsäuren mit population tagging - Google Patents

Vergleichende auswertung der nukleinsäuren mit population tagging

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Publication number
EP1360333A2
EP1360333A2 EP02720894A EP02720894A EP1360333A2 EP 1360333 A2 EP1360333 A2 EP 1360333A2 EP 02720894 A EP02720894 A EP 02720894A EP 02720894 A EP02720894 A EP 02720894A EP 1360333 A2 EP1360333 A2 EP 1360333A2
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EP
European Patent Office
Prior art keywords
nucleic acid
domain
sample
differentiation
target
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP02720894A
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English (en)
French (fr)
Inventor
Matthew M. Winkler
David Brown
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Asuragen Inc
Original Assignee
Ambion Inc
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Publication date
Application filed by Ambion Inc filed Critical Ambion Inc
Publication of EP1360333A2 publication Critical patent/EP1360333A2/de
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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
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection

Definitions

  • the present invention relates generally to the fields of nucleic acid amplification. More particularly, it concerns using nucleic acid amplification to compare two or more nucleic acid populations.
  • the present invention incorporates methods for adding nucleic acid tag sequences to nucleic acid populations to promote amplification and differentiation of one or more nucleic acid targets present in the nucleic acid population(s).
  • Gene expression analysis is the study of how much protein gets synthesized in a cell or tissue from a defined set of genes. The identity and abundance of proteins in a sample determines the type and state of the cell, tissue, organ or organism from which it derived. Unfortunately, the quantitative assessment of many different proteins in a given biological sample is exceedingly difficult and requires large amounts of sample.
  • RNAs in a sample can reveal which proteins are being expressed in a biological sample and at what levels.
  • the study of RNA expression is often easier than that of protein expression, thus RNA analysis is preferred by investigators studying the dynamics of gene expression.
  • Techniques commonly used for RNA expression analysis can be divided into those aimed at quantifying one or a few RNA targets in a sample and those designed to screen a large number of RNA targets in a sample.
  • Techniques for analyzing one or a few RNA targets include
  • Northern blot analysis has three shortcomings. First, the method is labor intensive. The process of fractionating RNA samples, transferring to membranes, generating probes for analysis, hybridizing probe to the Northern blot, and detecting hybridized probe requires several days to complete and numerous independent reagents. Second, Northern blot analysis is incapable of detecting rare messages. In general, 100,000 to 1,000,000 target molecules must be present in a sample for it to be detected via northern blotting. This tends to limit Northern blotting to the analysis of moderately and highly abundant RNA targets. Third, the method is typically limited to detecting a single target per hybridization reaction. For multiple targets to be assessed in a single hybridization experiment, the desired RNA targets must be of significantly different sizes and similar abundance. These two criteria are rarely met by multiple RNA targets.
  • Nuclease Protection Assay Another method of RNA expression analysis is the nuclease protection assay.
  • nuclease protection assay There are two types of nuclease protection assay, the SI assay and the ribonuclease protection assay (RPA), which differ primarily in the nuclease used to digest the samples being assayed (Sambrook, 1989).
  • the SI Assay uses Nuclease SI while RPA typically uses RNase A and/or RNase Tl. Both methods use labeled nucleic acid probes that are complementary to specific RNA targets in a sample. The labeled probes are incubated with RNA samples to allow hybridization to occur between the target RNA and labeled probe.
  • the mixture is then treated with one or more of the nucleases described above, each of which specifically degrades single- stranded RNA and/or DNA. Any labeled probe that is not hybridized to target RNA is degraded, leaving only the hybridized probe.
  • the undigested probe is fractionated by gel electrophoresis and visualized. The signal from the undigested probe can be quantified to determine the amount of target RNA in the samples being assessed.
  • Relative RT-PCR is far more sensitive than Northern analysis and nuclease protection assays.
  • the technique is easier to set up than the above methods because no probes need be synthesized for analysis.
  • the technique requires a great deal of effort to ensure that the amplification reaction is in linear range at the point that the amplification products are assessed.
  • the method is only relatively quantitative which means that it can help determine if a particular transcript is present at greater or lesser levels in one sample compared to another.
  • relative RT-PCR cannot reliably quantify the difference in the amount of RNA present in two samples.
  • RNA sample is aliquotted into tubes with differing amounts of competitor.
  • the RNA/competitor mixtures are reverse transcribed and amplified with primers specific to the target and competitor.
  • the mixture that results in equal amounts of amplification product for both the target and competitor reveals the concentration of the target in the sample.
  • Adaptor-Tagged Competitive-PCR is a variation of the competitive RT-PCR procedure that reduces the requirement for competitor synthesis and increases the number of samples that can be assessed in a single reaction (Kato 1997, European Patent
  • ATAC-PCR makes one sample population a competitor for another sample population. ATAC-PCR accomplishes this by converting mRNA samples to double- stranded cDNA using a reverse transcriptase, digesting the cDNA samples with a restriction enzyme, and ligating adapters to members of the cDNA samples at their respective restriction sites.
  • the adapters share a primer binding site but differ in size or sequence (i.e., unique restriction or hybridization sites).
  • the adapter-tagged cDNAs are mixed and amplified with a gene-specific primer and a PCR primer specific to the shared adapter sequence present at the proximal ends of the cDNA populations.
  • the amplification products resulting from PCR are directly assessed by gel electrophoresis. If the adapters from the populations differ by a restriction site, then the amplification products are aliquoted into different restriction digestion reactions to cleave the tag sequences from amplification products derived from specific samples. The digestion products are then assessed by gel electrophoresis. Because the amplification products generated from each sample population are different sizes, they can be readily fractionated and quantified. The ratio of amplification products generated from each sample reflects the relative abundance of the target in each sample.
  • ATAC-PCR has four shortcomings. First, four steps are required to convert an RNA sample to a population that is ready for PCR amplification. If any of these steps vary between the samples being compared, inaccuracies will result. Thus inefficient or biased reverse transcription, second strand cDNA synthesis, restriction digestion, or adapter ligation can profoundly affect the data being generated. Second, ATAC-PCR initiates amplification with double-stranded nucleic acids that all possess a domain that is complementary to the adapter- specific primer. Therefore, target and non-target sequences are at least linearly amplified from the amplification domain of the adapter. This generates background that can affect quantitative analysis. Third, ATAC-PCR is apparently limited to the comparative analysis of targets in only a few samples.
  • U.S. Patent 5,712,126 estimates that approximately 80% of the amplification products that appear to be differentially expressed in a DD-RT-PCR experiment turn out not to differ in relative expression level. U.S. Patent 5,712,126 also indicates that when a single RNA sample is split and the two resulting samples are taken through the DD-RT-PCR procedure, the fingerprint patterns differ by 5%. The inconsistency in generating fingerprints has kept the technique from becoming a preferred method for comparing RNA or DNA samples.
  • RNAs can be labeled during transcription and used directly for array analysis, or unlabeled aRNA can be reverse transcribed with labeled dNTPs to create a cDNA population for array hybridization. In either case, the detection and analysis of labeled targets is the same as described above.
  • aRNA amplification provides a way to assess small RNA samples, it is not yet clear that the amplification scheme is appropriate for comparative analysis. One potential problem is that amplification may be biased.
  • RNA populations being compared are assessed separately so that amplification products from each sample can be readily distinguished.
  • DD-RT-PCR for example, the RNA populations being compared are amplified in different reaction vessels and assessed by electrophoresis in adjacent lanes on an acrylamide gel.
  • the invention relates to methods of comparing one or more nucleic acid targets within two or more samples, comprising:
  • the nucleic acid tags may further comprise at least one additional domain of the type described elsewhere in the specification, for example, a labeling domain, a restriction enzyme domain, a secondary amplification domain, a secondary differentiation domain or a sequencing primer binding domain.
  • Some specific methods of the invention comprise comparing one or more nucleic acid targets within two or more samples, comprising:
  • the invention relates to methods of comparing one or more nucleic acid targets within two or more samples, comprising:
  • the differentiating further comprises annealing at least a second differentiation primer to the second primer binding domain, wherein the differentiating further comprises extension of the second differentiation primer to produce at least a second differentiated nucleic acid;
  • a or “an” may mean one or more.
  • the words “a” or “an” when used in conjunction with the word “comprising”, the words “a” or “an” may mean one or more than one.
  • another may mean at least a second or more.
  • a “plurality” means "two or more.”
  • FIG. 6 Quantitative analysis using size differentiation domains.
  • the present invention provides simple procedures for directly comparing single or multiple nucleic acid targets in two or more samples. By a process called
  • the invention differs from ATAC-PCR in several manners.
  • the present invention requires only a single step to tag a nucleic acid population. This reduces the likelihood that inaccuracies will result from variable reaction efficiencies.
  • ATAC-PCR requires four independent enzymatic reactions to tag a nucleic acid population which greatly increases the chances of sample-to-sample variability that can create quantitative aberrations in the experimental data.
  • tagged nucleic acids are single-stranded and require the action of a target specific primer to initiate amplification.
  • ATAC-PCR initiates amplification with double-stranded nucleic acids that all possess a domain that is complementary to the adapter-specific primer.
  • Embodiments of the present invention involve nucleic acids in many forms.
  • Nucleic acid samples are collections of RNA and/or DNA derived or extracted from chemical or enzymatic reactions, biological samples, or environmental samples.
  • Nucleic acid tags are nucleic acids of a defined sequence that are appended to nucleic acids in a sample to facilitate its analysis. There are many potential types of tags for use in the invention, which are described elsewhere in this specification.
  • nucleic acid is well known in the art.
  • a “nucleic acid” as used herein will generally refer to a molecule (i.e., a strand) of DNA, RNA or a derivative or analog thereof, comprising a nucleobase.
  • a nucleobase includes, for example, a naturally occurring purine or pyrimidine base found in DNA (e.g., an adenine "A,” a guanine “G,” a thymine “T” or a cytosine “C”) or RNA (e.g., an A, a G, an uracil "U” or a C).
  • Preferredine and/or “pyrimidine” nucleobase(s) encompass naturally occurring purine and/or pyrimidine nucleobases and also derivative(s) and analog(s) thereof, including but not limited to, a purine or pyrimidine substituted by one or more of an alkyl, caboxyalkyl, amino, hydroxyl, halogen (i.e., fluoro, chloro, bromo, or iodo), thiol or alkylthiol moeity.
  • Preferred alkyl (e.g., alkyl, caboxyalkyl, etc.) moeities comprise of from about 1, about 2, about 3, about 4, about 5, to about 6 carbon atoms.
  • a purine or pyrimidine include a deazapurine, a 2,6-diaminopurine, a 5-fluorouracil, a xanthine, a hypoxanthine, a 8- bromoguanine, a 8-chloroguanine, a bromothymine, a 8-aminoguanine, a 8-hydroxyguanine, a 8- methylguanine, a 8-thioguanine, an azaguanine, a 2-aminopurine, a 5-ethylcytosine, a 5- methylcyosine, a 5-bromouracil, a 5-ethyluracil, a 5-iodouracil, a 5-chlorouracil, a 5- propyluracil, a thiouracil, a 2-methyladenine, a methylthioadenine, a N,N-diemethyladenine
  • Patent 5,886,165 which describes oligonucleotides with both deoxyribonucleotides with 3 '-5' intemucleotide linkages and ribonucleotides with 2'-5' intemucleotide linkages;
  • U.S. Patent 5,714,606 which describes a modified intemucleotide linkage wherein a 3 '-position oxygen of the intemucleotide linkage is replaced by a carbon to enhance the nuclease resistance of nucleic acids;
  • U.S. Patent 5,672,697 which describes oligonucleotides containing one or more 5' methylene phosphonate intemucleotide linkages that enhance nuclease resistance;
  • Patents 5,466,786 and 5,792,847 which describe the linkage of a substituent moeity which may comprise a drug or label to the 2' carbon of an oligonucleotide to provide enhanced nuclease stability
  • U.S. Patent 5,223,618 which describes oligonucleotide analogs with a 2 or 3 carbon backbone linkage attaching the 4' position and 3' position of adjacent 5-carbon sugar moiety to enhanced resistance to nucleases and hybridization to target RNA
  • U.S. Patent 5,470,967 which describes oligonucleotides comprising at least one sulfamate or sulfamide intemucleotide linkage that are useful as nucleic acid hybridization probe
  • peptide-based nucleic acid analog or "PENAM”, described in U.S. Patent Serial Nos. 5,786,461, 5891,625, 5,773,571, 5,766,855, 5,736,336, 5,719,262, 5,714,331, 5,539,082, and WO 92/20702, each of which is incorporated herein by reference.
  • Peptide nucleic acids generally have enhanced sequence specificity, binding properties, and resistance to enzymatic degradation in comparison to molecules such as DNA and RNA (Egholm et al, 1993; PCT/EP/01219).
  • a peptide nucleic acid generally comprises one or more nucleotides or nucleosides that comprise a nucleobase moiety, a nucleobase linker moeity that is not a 5-carbon sugar, and/or a backbone moiety that is not a phosphate backbone moiety.
  • nucleobase linker moieties described for PNAs include aza nitrogen atoms, amido and/or ureido tethers (see for example, U.S. Patent No. 5,539,082).
  • backbone moieties described for PNAs include an aminoethylglycine, polyamide, polyethyl, polythioamide, polysulfmamide or polysulfonamide backbone moiety.
  • a tag or other nucleic acid used in the invention may be made by any technique known to one of ordinary skill in the art, such as for example, chemical synthesis, enzymatic production or biological production.
  • a synthetic nucleic acid e.g., a synthetic oligonucleotide
  • Non-limiting examples of a synthetic nucleic acid include a nucleic acid made by in vitro chemical synthesis using phosphotriester, phosphite or phosphoramidite chemistry and solid phase techniques such as described in EP 266,032, incorporated herein by reference, or via deoxynucleoside H- phosphonate intermediates as described by Froehler et al, 1986 and U.S. Patent Serial No.
  • a non-limiting example of a biologically produced nucleic acid includes a recombinant nucleic acid produced (i.e., replicated) in a living cell, such as a recombinant DNA vector replicated in bacteria (see for example, Sambrook et al. 1989, inco ⁇ orated herein by reference).
  • the present invention also encompasses a nucleic acid that is complementary to a specific nucleic acid sequence.
  • another nucleic acid may refer to a separate molecule or a spatial separated sequence of the same molecule.
  • the organism may be a virus.
  • the virus may be, but is not limited to, a DNA virus, including but not limited to a ssDNA vims or a dsDNA vims; a DNA RNA rev transcribing vims; a RNA vims, including but not limited to a dsRNA vims, including but not limited to a -ve stranded ssRNA or a +ve stranded ssRNA; or an unassigned vims.
  • RNA samples comprising populations designed by the hand of man may also be generated and used as a standard against which another sample or subpopulation of target sequences could be compared.
  • the synthetic population can be used to accurately quantify one or more targets from one or more sample(s) if the concentrations of the synthetic nucleic acids are known.
  • a synthetic sample may comprise a collection of nucleic acids (e.g., RNA, cDNA or genomic DNA) from many different tissues, cells (e.g., cell cultures), or other samples that could provide an average population against which a sample, or subpopulation of target sequences, could be compared.
  • Primer binding sites for other types of amplification methods might also be used as amplification domains. Often such primer binding regions share similar characteristics with PCRTM primer binding sites, however the primers used for other amplification methods typically possess sequences 5' to the binding domain.
  • primers for 3SR and NASBA contain an RNA polymerase promoter sequence 5' to the priming site to support subsequent transcription. Because 3SR and NASBA are performed at relatively low temperature (37°C to 42°C), the primer binding regions can have much lower melting temperatures than those used for PCRTM.
  • a tag comprise at least one differentiation domain.
  • a differentiation domain comprises a sequence that can be used to identify the sample from which a particular amplified nucleic acid derives.
  • a differentiation domain may comprise a different affinity sequence for removing one or more labeled nucleic acid(s) unique to each sample population (e.g., input sample populations in a sample mixture, a different primer binding domain for labeled DNA synthesis, a different transcription domain for labeled RNA synthesis, a size differentiation domain, an additional domain described herein or as would be known to one of skill in the art (e.g., a restriction enzyme site) or combinations thereof.
  • a differentiation domain may comprise a promoter sequence (a "transcription domain") that binds an RNA polymerase to initiate transcription.
  • the resulting differentiated RNA e.g., a labeled RNA
  • an amplified population possessing promoter sequences can be transcribed in a reaction (e.g., an in vitro reaction) with one or more labeled nucleotides (radio- or non-isotopic-labeled NTPs) and an appropriate RNA polymerase to convert double-stranded nucleic acid amplification products into differentiated RNAs that can be used for comparative analysis.
  • tag sequences may be appended to sample nucleic acids by reverse transcription.
  • tagged cDNA populations can be conveniently generated by priming reverse transcription with oligonucleotides comprising a tag sequence at its 5' end and sequence complementary to RNAs in a sample at its 3' end. Hybridization of the primer to one or more targets in an RNA sample and subsequent reverse transcription yields cDNA with tag sequences at its 5 'end.
  • a single-stranded DNA (e.g. , cDNA) population may be diluted in a buffer appropriate for hybridization and polymerization, and hybridized to one or more tags comprising specific or random sequences at their 3' ends and amplification and differentiation domain at their 5' ends.
  • Addition of a DNA polymerase such as, for example, the klenow fragment of DNA polymerase I or Taq DNA polymerase, will extend a tag to create a tagged population of DNA segments.
  • a disadvantage of appending double-stranded tags to double-stranded nucleic acids is that primers specific to the amplification domain of the tag can bind and be extended from target and non-target molecules alike. Using restriction digestion and double-stranded tag ligation may create far greater background than the other methods described for tagging a nucleic acid target and is therefore a less preferred method for tagging populations. This is in contrast to other tagging methods described herein, whereby single-stranded tags are appended to single-stranded nucleic acids from the sample. In these embodiments, the amplification domain of the tag sequence only becomes a primer binding site when the target specific primer is extended during the amplification phase.
  • RNA template into hundreds and even thousands of RNA transcripts. While this level of amplification is orders of magnitude less than what is achieved by PCR, NASBA, and SDA, it could be sufficient for some embodiments of the present invention.
  • the labeled nucleic acids resulting from the extension of any non- differentiation primers would be as likely to derive from an unintended sample as an intended sample.
  • the labeled nucleic acid would therefore not be specific to a single input sample making the labeled nucleic acids incompatible with comparative analysis.
  • a transcription reaction with one or more labeled nucleotides e.g., isotopic- or non-isotopic-labeled NTPs
  • an appropriate RNA polymerase can be used to convert double-stranded templates into differentiated RNAs that can be used for comparative analysis.
  • the amplified products generated from target(s) in a sample mixture can be split into multiple transcription reactions specific to each transcription promoter. Transcription reactions inco ⁇ orating one or more labeled NTPs create labeled RNAs specific to each input sample. The labeled RNAs can be used to compare the abundance of targets in each of the nucleic acid samples.
  • cloning of amplified nucleic acids may be accomplished without the use of restriction digestion.
  • U.S. Patent 5,487,993 takes advantage of the activity of many thermostable polymerases whereby a non-templated dATP is attached to the 3' ends of PCR amplified nucleic acids.
  • the PCR amplified nucleic acids can be readily ligated into linearized vectors possessing single T overhangs at their 3' ends without restriction digestion of the amplified nucleic acids. It is contemplated that this method could be inco ⁇ orated into the present invention by providing a rapid method to clone the amplified nucleic acids.
  • Identifying differentiation domains that function equally well and that do not affect amplification efficiency is relatively straightforward where primer extension, affinity purification or digestion is being used for differentiation. In these cases, altering the identity of just a few nucleotides can provide effective differentiation (e.g., labeling specificity); rarely does altering a few bases within the differentiation domain affect amplification efficiency. In addition, because both methods use the same enzyme (i.e., a single DNA polymerase) for generating labeled nucleic acids from each of the unique tags, polymerization biases should not introduce variability.
  • the first nucleic acid target can be one of a plurality of nucleic acid targets, and the first and second populations can be part of a plurality of populations being analyzed.
  • FIG. 3 depicts one of the most common embodiments of the invention, in which the same nucleic acid target is comprised within two or more populations.
  • the thick lines in FIG. 3 represent the tag sequences.
  • the thin lines represent the sequences of the RNA and/or DNA populations in which one or more nucleic acid targets are comprised.
  • a first nucleic acid tag comprising a differentiation domain having a first primer binding domain (PBS#1) is appended to the nucleic acid target of a first nucleic acid population.
  • a second nucleic acid tag comprising a differentiation domain having a second primer binding domain (PBS#2) is appended to the nucleic acid target of a second nucleic acid population.
  • the differentiation domain of the second nucleic acid tag is different than the differentiation domain of the first nucleic acid tag.
  • FIG. 4 depicts the application of the invention to compare at least a first nucleic acid target within two or more populations.
  • a nucleic acid tag comprising a differentiation domain that is a first transcription domain (i.e., a T7 promoter) is appended to a first nucleic acid target of a first nucleic acid population.
  • a second nucleic acid tag comprising a differentiation domain that is a second transcription domain (i.e., a SP6 promoter) is appended to the first nucleic acid target of a second nucleic acid population.
  • the transcription domain forming the differentiation domain of the second nucleic acid tag is specific for a different polymerase than that in the differentiation domain of the first nucleic acid tag. Any form of promoter and polymerase combination may be used, and the T7 and SP6 promoters, while very useful in the invention, are not limiting.
  • the first nucleic acid target can be only one of a plurality of nucleic acid targets and the first and second populations may be only two members of a plurality of populations being analyzed. However, for the sake of clarity, only one target and two populations are shown in this figure.
  • RT-PCR is ideally suited for confirming and quantifying targets that appear to be differentially expressed.
  • Comparative RT-PCR comprises reverse transcribing different mRNA populations using anchored oligodT primers with identical primer binding sites at their 5' ends (amplification domains) and different length polynucleotide linkers between the primer binding site and oligodT that function as differentiation domains.
  • Two or more differentially tagged cDNA populations are mixed and amplified by PCR using one primer specific to the tags and one or more primer(s) specific to a gene(s) of interest.
  • the resulting amplified nucleic acids are differentiated by fractionation using gel electrophoresis. Because the appended tags are different sizes for the different populations, the amplified nucleic acids that result from different populations migrate differently in the gel. These differentiated nucleic acids are then quantified to provide the relative expression of the target(s) in each of the populations.
  • a specific example of this protocol is shown in FIG. 6.
  • nucleic acid populations 1 and 2 are tagged by reverse transcription using primers with identical Primer Binding Sites (PBS) and a promoter for T7 or SP6 RNA polymerase.
  • PBS Primer Binding Sites
  • the differentially tagged cDNAs are mixed and targets are amplified by PCR using one primer specific to the PBS of the tag and a collection of primers specific to targets.
  • the amplified sample is split into two transcription reactions, one with T7 RNA polymerase and Cy3 NTP and one with SP6 RNA polymerase and a Cy5 NTP.
  • the labeled RNAs can then be hybridized to a single array.

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EP02720894A 2001-01-31 2002-01-31 Vergleichende auswertung der nukleinsäuren mit population tagging Withdrawn EP1360333A2 (de)

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