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WO2003054224A2 - Procede et dispositif integre permettant de deceler des methylations de la cytosine - Google Patents

Procede et dispositif integre permettant de deceler des methylations de la cytosine Download PDF

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Publication number
WO2003054224A2
WO2003054224A2 PCT/DE2002/004507 DE0204507W WO03054224A2 WO 2003054224 A2 WO2003054224 A2 WO 2003054224A2 DE 0204507 W DE0204507 W DE 0204507W WO 03054224 A2 WO03054224 A2 WO 03054224A2
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WIPO (PCT)
Prior art keywords
dna
primers
amplification
solid phase
primer
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PCT/DE2002/004507
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German (de)
English (en)
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WO2003054224A3 (fr
Inventor
Kurt Berlin
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Epigenomics AG
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Epigenomics AG
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Publication date
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Priority to AU2002357968A priority Critical patent/AU2002357968A1/en
Priority to EP02791612A priority patent/EP1451359A2/fr
Publication of WO2003054224A2 publication Critical patent/WO2003054224A2/fr
Publication of WO2003054224A3 publication Critical patent/WO2003054224A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification

Definitions

  • the present invention describes a method for the detection of the methylation state of genomic DNA samples.
  • 5-Methylcytosine is the most common covalently modified base in the DNA of eukaryotic cells. For example, it plays a role in the regulation of transcription, in genetic imprinting and in tumorigenesis. The identification of 5-methylcytosine as a component of genetic information is therefore of considerable interest. However, 5-methylcytosine positions cannot be identified by sequencing because 5- Methylcytosine has the same base pairing behavior as cytosine. In addition, in the case of PCR amplification, the epigenetic information which the 5-methylcytosines carry is completely lost.
  • the state of the art in terms of sensitivity is defined by a method which includes the DNA to be examined in an agarose matrix, thereby preventing the diffusion and renaturation of the DNA (bisulfite only reacts on single-stranded DNA) and all precipitation and purification steps replaced by rapid dialysis (Olek, A. et al., Nucl. Acids. Res. 1996, 24, 5064-5066). With this method, individual cells can be examined, which illustrates the potential of the method. However, only individual regions up to about 3000 base pairs in length have so far been investigated, and a global examination of cells for thousands of possible methylation analyzes is not possible.
  • PCR reactions polymerase chain reactions
  • two specifically binding primers one complementary to the (+) strand, the other complementary to the (-) strand of the one to be amplified Templates is.
  • the aim of such an amplification is to reproduce a certain fragment of the Te plat DNA, which is usually precisely defined and is largely or completely known in its base sequence.
  • PCR reactions using more than two different primers are also known. In most cases, they are used for the amplification of several fragments, likewise at least largely known in their base sequence, simultaneously in one vessel. In this case too, the primers used specifically bind to certain sections of the template DNA.
  • the hybridization chamber (DE19952723) developed by Epigenomics AG carries out periodic denaturation of the DNA sample without generating a detachment of DNA already hybridized to oligomers. Because the DNA sample is thermally denatured before being applied to the array and then suddenly cooled, it is predominantly in single-stranded form when contacted with the array. This means that a large part of the otherwise double-stranded DNA sample is available for hybridizations with the oligomer array. By pumping the sample liquid back and forth, the device ensures that this process is repeated until a sufficient part of the double-stranded DNA sample has hybridized to the oligomers of the array. At the same time, this process causes mixing in the chamber during the hybridization phase.
  • Genomic DNA is obtained by standard methods from DNA from cell, tissue or other test samples. This standard methodology can be found in references such as Fritsch and Maniatis eds., Molecular Cloning: A Laboratory Manual, 1989.
  • Coded particles have been used in very different areas for some time. Color-coded beads have been used for the parallel diagnosis of T and B cells (Baran and Parker, Am. J. Clin. Pathol. 1985, 83, 182-9). Beads containing radiaoactive indium have been used as indicators of the motility of the gastrointestinal tract (Dormehl et al Eur. J. Nucl. Med. 1985, 10, 283-5). Companies such as Lu inex or Illumina, who run highly parallel diagnostics with color-coded plastic beads, use 100 different different -coloured beads, to which many different probes can be attached. As a result, 100 different parameters can be queried in a reaction, which could be, for example, 100 different diagnostic tests.
  • the present invention is intended to provide a method which is particularly suitable for the detection of DNA polymorphisms and cytosine methylations in genomic DNA samples.
  • a device for analyzing DNA samples should preferably be used, which enables both the amplification of DNA fragments and the analysis of DNA polymorphisms and cytosine methylations in a vessel by means of immobilized primers and probes.
  • the object is achieved according to the invention by a method for the detection of DNA polymorphisms and cytosine methylation, the following steps being carried out: a) the DNA to be examined is obtained from a sample; b) the DNA to be examined is amplified in a polymerase reaction, a primer being in solution and a primer being bound to the surface of a solid phase, and at least one of the primers consisting of two domains, the one located at the 3 'end the DNA to be examined hybridizes, while the one located at the 5 'end does not hybridize; c) the DNA amplified in step b) is amplified again, the primers of this second amplification the domain located at the 5 'end hybridize or are identical to at least one of the first primers and these primers are provided with a detectable label for the second amplification; d) the amplificates are on oligonucleotides and / or
  • PNA oligomers hybridized, which are bound to a solid phase, which do not act as primers in the amplification, and concluded from the hybridization on sequence features or methylation ester of the DNA to be examined.
  • step a) the DNA to be examined is treated chemically and / or enzymatically after it has been obtained.
  • a particularly preferred embodiment of the method according to the invention for the detection of DNA polymorphisms and cytosine methylation is characterized in that the following steps are carried out: the DNA to be examined is obtained from a sample;
  • the DNA to be examined is treated chemically and / or enzymatically;
  • the DNA to be examined is amplified in a polymerase action, a primer being in solution and a primer being bound to the surface of a solid phase and at least one of the primers consisting of two domains, the one located at the 3 'end being attached to the DNA to be examined hybridizes, while the one located at the 5 'end does not hybridize;
  • the DNA amplified in the previous step is amplified again, the primers of this second amplification hybridizing or being identical to at least one of the first primers to the domain located at the 5 'end, and these primers being provided with a detectable label for the second amplification are; -
  • the amplificates are hybridized to oligonucleotides and / or PNA oligomers, which are bound to a solid phase, which do not function as primers in the amplification, and conclude from the hybridization on sequence features or methylation patterns of the DNA to be examined.
  • primers of the first amplification (step b) and the oligonucleotides (step d) are immobilized on the same solid phase.
  • the solid phase is flat. Furthermore, it is particularly preferred according to the invention that the solid phase is made of glass, metal, in particular gold or plastic.
  • beads are used as the solid phase, each bead being coded differently. It is particularly preferred here that the beads use fluorescent dyes and absorbents
  • Dyes via chemiluminescence, via transponders, via nuclides or via chemical labels which can be detected by mass spectrometry.
  • the surface is chemically treated in such a way that the oligonucleotides and primers can be covalently bound to it.
  • the present invention furthermore relates to a device consisting of a) a surface on which primer oligonucleotides are immobilized with their 5 'end and additionally oligonucleotides and / or PNA olig eres which cannot be extended by a polymerase; b) a chamber, the surface of which is a wall of this Chamber forms; c) a temperature control unit that controls the chamber temperature; d) a system which supplies liquids to the chamber.
  • the DNA to be examined is obtained from a sample.
  • the genomic DNA to be analyzed is preferably obtained from the usual sources for DNA, such as. B. cell lines, blood, sputum, stool, urine, brain spinal fluid, paraffin-embedded tissue, for example tissue from the eyes, intestines, kidneys, brain, heart, prostate, lungs, chest or liver, histological slides and all possible combinations of these.
  • the DNA can also already be processed or isolated.
  • the DNA to be examined is treated chemically and / or enzymatically.
  • bisulfite disulfite, hydrogen sulfite
  • the enzymatic treatment is preferably carried out using methylation-sensitive restriction enzymes which can differentiate between methylated and unmethylated DNA.
  • the DNA to be examined is amplified in a polymer laser action, with a
  • Primer is in solution and a primer is bound to the surface of a solid phase and at least one of the primers consists of two domains, the domain located at the 3 'end hybridizing to the DNA to be investigated, while the domain located at the 5' end not hybridized.
  • the amplified sections are immobilized on a solid phase via at least one primer used in the amplification.
  • the primer is preferably bound by chemical reactions (e.g. by introducing a C6 5 'amino function).
  • the 3 'domain preferably contains only bases C, A and T or bases G, A and T, as corresponds to bisulfite-treated DNA. In the 5 'domain, however, all four bases A, C, G and T are preferably included.
  • primers consisting of two domains
  • two-stage amplifications can be carried out, which particularly specifically provide a large number of fragments at the same time and which at the same time solves the problem that a large number of labeled and correspondingly expensive primers are normally used for such a complex amplification Need to become.
  • the amplification of the DNA in the two previous method steps is preferably carried out in a polymer chain reaction, preferably with a heat-resistant DNA polymerase.
  • the amplification of several DNA sections is preferably done in a reaction vessel.
  • the PCR products are preferably labeled via absorbing dyes and / or via chemiluminescence and / or via radioactive isotopes and / or via fluorescent labels.
  • the fluorescent label by a fluorescence-labeled nucleotide such.
  • B Cy5-dCTP introduced.
  • the amplificates are hybridized to oligonucleotides and / or PNA oligomers which are bound to a solid phase, which do not function as primers in the amplification, and from the hybridization conclusions are drawn about sequence features or methylation patterns of the DNA to be examined.
  • the hybridized oligonucleotides can be provided at the 3 'end with the following modifications, for example: amino, phosphate, thiophosphate, dideoxy, carboxy, dihydroxy, O-acetyl or alkyl.
  • DNA polymorphisms and cytosine methylations are analyzed in one experiment.
  • the present invention also relates to a device for the in situ amplification and analysis of DNA samples, as shown in FIG. 4. It consists of a closed and temperature-controlled hybridization chamber, a pump, a heating element and a cooling element, each of which is connected to one another by liquid-conveying conduction paths, preferably plastic tubes (DE 19952723 AI) and a commercially available slide (oligonucleotide array) on which DNA oligomers which can act as primers in a polymerase action, and PNA oligomers or DNA oligomers which cannot act as primers in a polymerase action are arranged.
  • the volume that the hybridization chamber holds when an oligomer array is inserted is particularly preferably less than 200 ⁇ l.
  • the present invention furthermore relates to a device for analyzing DNA samples.
  • This consists of at least one surface on which DNA oligomers which can act as primers in a polymerase action and PNA oligomers or DNA oligomers which cannot act as primers in a polymerase action are arranged.
  • the particularly preferred pumping of the sample liquid back and forth in the device ensures that a sufficient part of the double-stranded DNA sample hybridizes to the oligomers of the array.
  • ESR1 Acc. No. EP11141
  • PCR conditions 2 ⁇ l DNA (20 ng) (bisulfite treated); 0.4 ⁇ l Taq (2 units, Qiagen); 0.4 ⁇ l dNTP (25 each mmol / 1, MBI Fermentas) 0.25 mmol / 1 final concentration; 2 ul
  • Primer2 (12.5 pmol / ⁇ l) 0.5 pmol / ⁇ l final concentration; 5 ul 10X PCR buffer (Qiagen); 1 ⁇ l MgCl (15 mmol / 1, Qiagen)
  • the purified PCR products were used for the immobilization on glass surfaces (beads). They only differed in their 5 'modification. One product had the H 2 group at the 5 'end of the upper strand (for5 ward), the other had this group at the 5' end of the lower strand. reverse strand. During the test, double strands were bound, but after denaturing and washing, two different single strands are immobilized. The success of this binding process was determined using fluorescence measurements. First, a fluorescence signal from the beads was detected. Secondly, a decrease in the fluorescence signal of the immobilization buffer compared to a control buffer could be shown.
  • the beads were washed several times in the reaction vessels under denaturing conditions. After each denaturation step, the beads were additionally washed with 1 ml of water and some beads of each type were kept. All other beads were used for the next denaturation step (1. 5 min, 95 ° C with water; 2. 10 min, 25 ° C with 10 mM P0 4 buffer / 1% SDS; 3. 2 times 5 min, 25 ° C with 0.05 M NaOH; 4. 30 min 0.2 M NaOH / 0.1% SDS) was used. At the end the beads were dried in a vacuum and stored.
  • the DNA immobilized on the glass surfaces which was washed 4 times and denatured (see above), was used as a template for the PCR.
  • a master mix was prepared for the PCR and only one bead was added to each reaction vessel.
  • PCR conditions for 1 glass bead or 10 ng bisulfite-treated DNA as a control 0.2 ⁇ l Taq (1 unit), 0.2 ⁇ l dNTP (each 25 mM) 0.2 mM final concentration, 1 ⁇ l primer (12.5 pmol / ⁇ l) 0.5 pmol / ⁇ l final concentration; 1 ul Primer2 (12.5 pmol / ⁇ l) 0.5 pmol / ⁇ l final concentration; 2.5 ul (10x) buffer; 20.1 ul H 2 0
  • Example 2 Primer extension of an immobilized ESRI primer and amplification of the immobilized extension product with gene-specific and generic (M13 domain) PCR primers.
  • the immobilization buffer was optimized for the application of NH 2 -labeled oligonucleotides to PITC-derivatized lysine surfaces.
  • PCR was carried out in 0.2 ml reaction tubes as described above in the Eppendorf cycler; 5 primer-loaded PITC beads were placed in each reaction tube; PCR product was used as template (purified, undiluted, 5'-Cy5 labeled):
  • the primers of ESRI and MDRI with amino function were immobilized on PITC beads. All primers have an M13 domain and a gene-specific domain. After the immobilization, the beads were added to the PCR preparation without additional primer oligonucleotides using a PCR product as a template. During the cycles, the bound primers are attached to single-stranded DNA of the PCR products and extended. The success of the extension reaction is checked with this PCR. ESRI PCR product was used as a template that has no M13 domain. The primers used are specific for the extended primers (see results below). At a extension on the glass surfaces, only PCRs 3a and 4b are positive, as are controls 7a and
  • Example 3 In situ amplification of the ESRI gene and analysis of the methylation status of a CpG position on a microarray.
  • a glass slide (array) was chemically modified and the following oligonucleotides with a commercially available
  • ESRI-CG TAGGTTTTCGGGGTAGGG
  • ESR1-TG TAGGTTTTTGGGGTAGGG
  • the distribution of the oligonucleotides on the array is shown in FIG. 5.
  • the in-situ amplification was carried out in the hybridization chamber developed by Epigenomics AG.
  • 200 ⁇ l of the PCR solution 50 ng bisulfite-treated human genomic DNA, dNTPs 0.25 mM, 0.5 ⁇ M primer ESRI- BL ACAATAAAACCATCCCAAATAC or primer ESR1-BU-Ml3b CAGGAAACAGCTATGACACAATAAAACCATCCCAAATAC, 20 ⁇ l lOx reaction buffer, 5 U Taq polymerase (Qiagen, Hilden) filled in a reaction vessel (volume 500 ⁇ l) and covered with mineral oil.
  • the reaction vessel was placed in the denaturing position of the hybridization chamber and connected to the reaction (hybridization) chamber with a Teflon tube (see FIG. 4).
  • the PCR reaction was carried out by pumping the PCR solution back and forth between the denaturation position and the reaction (hybridization) chamber. This process was controlled by the following pump / temperature program (Tab. 1, steps 1-8). The PCR reaction was finished after the program steps 2-7 had been run through 38 times.
  • Step duration T-chamber pump T-Denat. Position d. PCR solution.
  • step 8 After the in-situ amplification (step 8, table 1), 400 ⁇ l hybridization buffer (6.9 x SSC, 2.7 Na-
  • Fig. 1 ESR PCR product
  • reaction vessels (Bead No. B): B1: beads after the second denaturation; B2: beads after the 3rd denaturation; B3: beads after the 4th denaturation; B4: no beads, no template;
  • Fig. 5 Schematic representation of the chip layout:
  • the black dots represent the positions of the immobilized primers (B). The location of the
  • Methylation detection oligos on the chip are represented by white dots TG oligonucleotide (TAGGTTTTTGGGGTAGGG) and gray dots CG oligonucleotide (TAGGTTTTCGGGGTAGGG) (A).

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Abstract

La présente invention concerne un procédé permettant de déceler des polymorphismes de l'ADN et une méthylation de la cytosine. Ce procédé consiste a) à extraire l'ADN à analyser d'un prélèvement; b) à traiter par voie chimique et/ou enzymatique l'ADN à analyser si une analyse de la méthylation de la cytosine est à effectuer ; c) à amplifier l'ADN à analyser dans une réaction de polymérase, une amorce étant présente dans la solution et une amorce étant liée à la surface d'une phase solide, au moins une de ces amorces étant constituée de deux domaines, celui situé à l'extrémité 3' étant hybridé à l'ADN à analyser mais celui situé à l'extrémité 5' n'étant pas hybridé ; d) à procéder à une nouvelle amplification de l'ADN amplifié en c), l'amorce de cette deuxième amplification étant hybridée au domaine situé à l'extrémité 5' d'au moins une première amorce ou lui étant identique, ces amorces de la deuxième amplification étant dotées d'une marque décelable ; e) à hybrider les produits d'amplification aux oligonucléotides et/ou aux oligomères PNA qui sont liés à une phase solide et ne fonctionnent pas comme amorces dans l'amplification. L'hybridation permet de tirer des conclusions sur les caractéristiques de séquence ou le modèle de méthylation de l'ADN à analyser.
PCT/DE2002/004507 2001-12-05 2002-12-05 Procede et dispositif integre permettant de deceler des methylations de la cytosine Ceased WO2003054224A2 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU2002357968A AU2002357968A1 (en) 2001-12-05 2002-12-05 Method and integrated device for the detection of cytosine methylations
EP02791612A EP1451359A2 (fr) 2001-12-05 2002-12-05 Procede et dispositif integre permettant de deceler des methylations de la cytosine

Applications Claiming Priority (2)

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DE10160983.3 2001-12-05
DE10160983A DE10160983B4 (de) 2001-12-05 2001-12-05 Verfahren und Integrierte Vorrichtung zum Nachweis von Cytosinmethylierungen

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005204652A (ja) * 2003-12-16 2005-08-04 Bayer Healthcare Llc メチル化特異的プライマー伸長（ｍｓｐｅ）によるメチル化状況を検出するアッセイ
EP1525328B1 (fr) * 2002-08-02 2010-01-20 Epigenomics AG Procede d'amplification d'acides nucleiques de faible complexite

Families Citing this family (2)

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Publication number Priority date Publication date Assignee Title
WO2007032748A1 (fr) * 2005-09-15 2007-03-22 Agency For Science, Technology & Research Procede de detection de la methylation de l'adn
AT502549B1 (de) 2005-10-07 2007-06-15 Anagnostics Bioanalysis Gmbh Vorrichtung zur analyse von flüssigen proben

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US5629158A (en) * 1989-03-22 1997-05-13 Cemu Bitecknik Ab Solid phase diagnosis of medical conditions
WO1993004199A2 (fr) * 1991-08-20 1993-03-04 Scientific Generics Limited Procedes de detection et de quantification d'acides nucleiques, et de production d'acides nucleiques marques, immobilises
CA2122450C (fr) * 1991-11-01 2004-07-13 Charles Phillip Morris Processus d'amplification en phase solide
US6310354B1 (en) * 1996-12-03 2001-10-30 Erkki Soini Method and a device for monitoring nucleic acid amplification reactions
DE60040741D1 (de) * 1999-04-20 2008-12-18 Illumina Inc Detektion von nukleinsäurereaktionen auf kügelchen-arrays
DE19934084A1 (de) * 1999-07-15 2001-01-18 Universitaetsklinikum Charite Verfahren und Testbesteck zur Markierung von DNA-Fragmenten während einer PCR-Reaktion
DE19935772C2 (de) * 1999-07-26 2002-11-07 Epigenomics Ag Verfahren zur relativen Quantifizierung der Methylierung von Cytosin Basen in DNA-Proben
DE19952723C2 (de) * 1999-10-26 2002-10-31 Epigenomics Ag Vorrichtung und Verfahren zur Hybridisierung doppelsträngiger DNA-Proben an Oligomer-Arrays
EP1255860A2 (fr) * 1999-12-29 2002-11-13 Mergen Ltd. Procedes d'amplification et de detection de plusieurs polynucleotides sur un support en phase solide
DE10010280B4 (de) * 2000-02-25 2006-08-10 Epigenomics Ag Verfahren zur Detektion von Cytosin-Methylierung in DNA Proben

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1525328B1 (fr) * 2002-08-02 2010-01-20 Epigenomics AG Procede d'amplification d'acides nucleiques de faible complexite
JP2005204652A (ja) * 2003-12-16 2005-08-04 Bayer Healthcare Llc メチル化特異的プライマー伸長（ｍｓｐｅ）によるメチル化状況を検出するアッセイ

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WO2003054224A3 (fr) 2003-10-30
AU2002357968A1 (en) 2003-07-09
DE10160983B4 (de) 2004-12-09
EP1451359A2 (fr) 2004-09-01
DE10160983A1 (de) 2003-06-26

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