EP1238112A2 - Procede de detection parallele de l'etat de methylation d'adn genomique - Google Patents

Procede de detection parallele de l'etat de methylation d'adn genomique

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Publication number
EP1238112A2
EP1238112A2 EP00989842A EP00989842A EP1238112A2 EP 1238112 A2 EP1238112 A2 EP 1238112A2 EP 00989842 A EP00989842 A EP 00989842A EP 00989842 A EP00989842 A EP 00989842A EP 1238112 A2 EP1238112 A2 EP 1238112A2
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EP
European Patent Office
Prior art keywords
factor
dna
binding protein
primers
transcription factor
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.)
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EP00989842A
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German (de)
English (en)
Inventor
Alexander Olek
Christian Piepenbrock
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Epigenomics AG
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Epigenomics AG
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Publication of EP1238112A2 publication Critical patent/EP1238112A2/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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to a method for the parallel detection of the methylation state of genomic DNA.
  • the present invention describes a method for parallel detection of the methylation state of genomic DNA samples, starting from a sample simultaneously amplifying numerous different fragments from sequences involved or / and transcribed and / or translated sequences and then the sequence context contained in the amplified fragments of CpG Dinucleotides is examined.
  • 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, genomic 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 since 5-methylcytosine has the same base pairing behavior like cytosine. In addition, in the case of PCR amplification, the epigenetic information which the 5-methylcytosines carry is completely lost. The modification of the genomic base cytosine to 5'-methylcytosine represents the most important and best-studied epigenetic parameter to date. Nevertheless, there are still methods to determine comprehensive genotypes of cells and individuals, but no comparable approaches to a large extent Generate and evaluate epigenotypic information.
  • REs restriction endonucleases
  • methylation-sensitive restriction endonucleases
  • REs are characterized by the fact that they cut a DNA into a specific DNA sequence, usually between 4 and 8 bases long The position of such sections can then be verified by gel electrophoresis, transfer to a membrane and hybridization.
  • Methylation-sensitive means that certain bases within the recognition sequence must be unmethylated in order for the section to be carried out according to the methylation pattern of the DNA
  • the fewest methylable CpG are within the recognition sequences of REs and cannot be examined with this method.
  • PCR combines a variant with this method; amplification by two primers located on both sides of the recognition sequence takes place after a cut only if the recognition sequence is methylated.
  • the sensitivity increases theoretically to a single molecule of the target sequence, but only individual positions can be examined with great effort (Shemer, R. et al., PNAS 93, 6371-6376). Again, it is a prerequisite that the methylable position is within the recognition sequence of a RE.
  • the second variant is based on partial chemical cleavage of total DNA, following the example of a Maxam-Gilbert sequencing reaction, ligation of adapters to the ends generated in this way, amplification with generic primers and separation on a gel electrophoresis. With this method, defined areas up to the size of less than a thousand base pairs can be examined. However, the process is so complicated and unreliable that it is practically no longer used (Ward, C. et al., J. Biol. Chem. 265, 3030-3033).
  • a relatively new and the most frequently used method for the investigation of DNA for 5-methylcytosine is based on the specific reaction of bisulfite with cytosine, which is converted into uracil after subsequent alkaline hydrolysis, which corresponds to the thymidine in its base-pairing behavior.
  • 5-methylcytosine is not modified under these conditions.
  • the original DNA is thus converted in such a way that methylcytosine, which originally cannot be distinguished from the cytosine by its hybridization behavior, can now be detected by "normal" molecular biological techniques as the only remaining cytosine, for example by amplification and hybridization or sequencing.
  • 5-methylcytsosine can also be found in the following review article: Rein, T., DePamphilis, M. L, Zorbas, H., Nucleic Acids Res. 26, 2255 (1998).
  • MAR matrix attachment regions
  • SAR scaffold attachment regions
  • insulators can, for example, inhibit the effect of the enhancer on the promoter if they are located between the enhancer and the promoter or, if they are located between heterochromatin and a gene, protect the active gene from the influence of the heterochromatin.
  • Examples of such insulators are: 1. So-called LCR (locus control regions), which consists of several sites that are hypersensitive to DNAase I; 2. Certain sequences such as SCS (specialized chromatin structures) or SCS ', 350 or 200 bp long and highly resistant to degradation by DNAase I and flanked on both sides by hypersensitive sites (100 bp spacing). The protein BEAF-32 binds to scs'. These insulators can lie on both sides of the gene.
  • Patents related generally to the use of oligomer arrays and photolithographic mask design include e.g. B. US-A 5,837,832, US-A 5,856,174, WO-A 98/27430 and US-A 5,856,101.
  • Patents that restrict the use of photolabile protective groups on nucleosides such as. B. WO-A98 / 39348 and US-A 5,763,599.
  • Matrix-assisted laser desorption / ionization mass spectrometry is a new, very powerful development for the analysis of biomolecules (Karas, M. and Hillenkamp, F. 1988. Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal. Chem 60: 2299-2301).
  • An analyte molecule is embedded in a UV absorbing matrix.
  • a short laser pulse evaporates the matrix into a vacuum, thus transporting the analyte unfragmented into the gas phase.
  • An applied voltage accelerates the ions into a field-free flight tube. Due to their different masses, ions are accelerated to different extents. Smaller ions reach the detector earlier than larger ones and the flight time is in the mass of the Converted ions.
  • Fluorescent-labeled probes have been used in many cases for scanning an immobilized DNA array.
  • the simple application of Cy3 and Cy5 dyes to the 5'OH of the respective probe is particularly suitable for fluorescence labeling.
  • the fluorescence of the hybridized probes is detected, for example, using a confocal microscope.
  • the dyes Cy3 and Cy5, among many others, are commercially available.
  • DNA base frequency counts indicate that the four bases in the DNA are not equally distributed. Accordingly, the following frequencies (probabilities of occurrence) of the bases can be determined from DNA databases.
  • Model 1 can be improved with the help of these values.
  • P 2s (PrimD) P b ⁇ NA ( ⁇ * P bDNA (A) '* P bDNA (CY * P büNA (G) c (model 3 for bisulfite DNA strand)
  • P 2a (PrimD) P b ⁇ NA (A r * P bDNA (T) '* P bDNA (G) s * P b ⁇ NA (c ⁇ (model 3 for anti-sense strand to a bisulfite DNA strand)
  • Model 3 takes this property into account and assumes that the primary DNA is a random sequence with dependence on directly adjacent bases (Markov chain of the first order).
  • the pairwise base probabilities determined empirically from the database result for both DNA strands as P bDNA (from; to) from the following table:
  • P 3s (PrimE) P rbDNA (B) P rbDNA ( B ⁇ • B l) P rbDNA ( B 2> 'ß l) P ' rbDNA ( B 3> 'B ⁇ )
  • the bisulfite treated DNA is amplified using a number of primers. From the point of view of the model, the DNA consists of one sense and one anti-sense strand with a length of N bases (all chromosomes are summarized here). A primer can be expected to be on the sense strand
  • This method provides a precise expectation for predicting the number of binding sites of certain sequences to any genomic DNA fragment previously treated with bisulfite. It serves as the basis for the calculation of the statistically expected number of amplificates in a PCR reaction based on two primer sequences and a DNA of length N, whereby only the amplificates that do not exceed a number of M nucleotides are taken into account.
  • M is 2000.
  • the known methods for the detection of cytosine methylations in genomic DNA are not designed in such a way that a large number of target regions in the genome to be examined are detected simultaneously.
  • the object of the present invention is to create a method with which a sample of genomic DNA can be examined simultaneously at several positions for cytosine methylation.
  • target regions can be amplified simultaneously using appropriately adapted primer pairs. It is not absolutely necessary to know the sequence context of all these target regions in advance, since in many cases, as also exemplified below, consensus sequences from the sequencing of related target regions are known, which, as described below, are used for the design of specific target regions of specific or selective primer pairs can be.
  • the method is successfully applied when the amplification of the chemically pretreated genomic DNA yields more fragments up to a maximum of 2000 base pairs in length than can be statistically expected from the target regions to be examined in each case.
  • the statistical expected value for the number of these fragments is calculated using the formulas listed in the prior art.
  • the number of fragments produced in the amplification step can be detected by any molecular biological, chemical or physical method.
  • the human haploid genome contains 3 billion base pairs and 100,000 genes, which in turn code an average of 2,000 base pairs long mRNA, the genes including the introns are on average 15,000 base pairs long. Promoters cover an average of 1000 base pairs per gene. If the statistical expected value for the number of amplified products that are based on two primers in transcribed sequences must therefore be calculated, the expected value for the entire genome must first be calculated using the above formula (method 3) and with the proportion of the transcribed sequences in the total genome to calculate. The same procedure is used for parts of any genome as well as for promoters and translated sequences (coding for mRNA).
  • the present invention thus describes a method for the parallel detection of the methylation state of genomic DNA. Several cytosine methylations in a DNA sample are to be analyzed simultaneously. The following process steps are carried out one after the other:
  • genomic DNA sample is chemically treated in such a way that at the 5'-position unmethylated cytosine bases are converted into uracil, thymine or another base which is unlike the cytosine in hybridization behavior.
  • the treatment of genomic DNA with bisulfite (hydrogen sulfite, disulfite) and subsequent alkaline hydrolysis, which leads to a conversion of unmethylated cytosine nucleobases into uracil, is preferably used for this purpose.
  • a second process step more than ten different fragments are simultaneously amplified from the pretreated genomic DNA by using synthetic oligonucleotides as primers, whereby more than twice as many fragments as statistically expected come from sequences involved in transcription and / or translation that are involved in gene regulation. This can be achieved using various methods.
  • At least one of the oligonucleotides used for the amplification contains fewer nucleobases than statistically a sequence-specific hybridization to the chemically treated genomic DNA sample would be required, which can lead to the amplification of several fragments at the same time.
  • the total number of nucleobases contained in this oligonucleotide is less than 17. In a particularly preferred variant of the method, the number of nucleobases contained in this oligonucleotide is less than 14.
  • oligonucleotides with different sequences are used simultaneously in one reaction vessel for the amplification.
  • more than 26 different oligonucleotides are used simultaneously to produce a complex amplificate.
  • more than twice the number that is statistically to be expected comes from genome sections involved in the regulation of genes, e.g. Promoters and enhancers, comes as would be expected with a purely random choice of the oligonucleotide sequences.
  • more than twice the number of the amplified fragments originates from genome sections which are transcribed in mRNA in at least one cell of the respective organism, or from genome sections (exons) spliced into mRNA after transcription than would be expected if the oligonucleotide sequences were chosen at random.
  • more than twice the number of the amplified fragments comes from genome sections which code for parts of one or more gene families, or else they come from genome sections which are used for so-called “matrix attachment sites” (MARs).
  • MARs matrix attachment sites
  • more than twice as many of the amplified fragments come from genome sections which organize the packing density of the chromatin as so-called “boundary elements”, or else they come from multiple drug resistance genes (MDR) - Promoters or coding regions than would be expected with a purely random choice of the oligonucleotide sequences.
  • MDR drug resistance genes
  • two oligonucleotides or two classes of oligonucleotides are used to amplify the fragments described, one or a class of which, except in the context of CpG or CpNpG, may contain base C but not base G and which the other or the other class may contain the base G, but not the base C, except in the context of CpG or CpNpG.
  • the amplification is carried out by means of two oligonucleotides, one of which contains a four to sixteen base long sequence which is complementary to or corresponds to such a DNA as it would arise if an equally long DNA fragment, which one of the factors
  • Amt aryl hydrocarbon receptor nuclear translocator AML-1a CBFA2 core binding factor, runt domain, alpha subunit 2
  • AP-1 activator protein-1 (AP-1); Synonyms: c-Jun
  • CDP CUTL1 cut (Drosophila) -Iike 1 (CCAAT displacement protein)
  • CDP CUTL1 cut (Drosophila) -Iike 1 (CCAAT displacement protein)
  • CDP CR1 complement component (3b / 4b) receptor 1
  • CDP CR3 complement component (3b / 4b) receptor 3 CHOP-C / EBPalpha DDIT; DNA-damage-inducible transcript 3 / CCAAT / enhancer binding protein (C / EBP), alpha c-Myc / Max avian myelocytomatosis viral oncogene / MYC-ASSOCIATED
  • CRE-BP1 / c-Jun activator protein-1 (AP-1); Synonyms: c-Jun CREB MP responsive element binding protein E2F E2F transcription factor (originally identified as a DNA- binding protein essential E1A-dependent activation of the adenovirus E2 promoter)
  • E47 transcription factor 3 E2A immunoglobulin enhancer binding factors E12 / E47
  • E47 transcription factor 3 E2A immunoglobulin enhancer binding factors E12 / E47
  • Egr-1 early growth response 1 Egr-2 early growth response 2 (Krox-20 (Drosophila) homolog)
  • ELK-1 ELK1 member of ETS (environmental tobacco smoke) oncogene family
  • HNF-1 TCF1 transcription factor 1, hepatic; LF-B1, hepatic nuclear factor (HNF1), albumin proximal factor
  • IRF-1 interferon regulatory factor 1 IRF-1 interferon regulatory factor 1
  • MEF-2 MADS box transcription enhancer factor 2, polypeptide A (myocyte enhancer factor 2A)
  • MZF1 ZNF42 zinc finger protein 42 (myeloid-specific retinoic acid-responsive)
  • MZF1 ZNF42 zinc finger protein 42 (myeloid-specific retinoic acid-responsive)
  • NF-E2 NFE2 nuclear factor (erythroid-derived 2), 45kD NF-kappaB (p50) nuclear factor of kappa light polypeptide gene enhancer in B-cells p50 subunit
  • P53 tumor protein p53 (Li-Fraumeni syndrome); TP53
  • Pax-3 paired box gene 3 (Waardenburg syndrome 1)
  • Pax-6 paired box gene 6 aniridia, keratitis
  • SRF serum response factor c-fos serum response element-binding transcription factor
  • TATA cellular and viral TATA box elements Tax / CREB Transiently-expressed axonal glycoprotein / cAMP responsive element binding protein
  • TCF11 Transcription Factor 11; TCF11; NFE2L1; nuclear factor
  • YY1 ubiquitously distributed transcription factor belonging to theGLI-Kruppel class of zinc finger proteins
  • binds would be treated chemically in such a way that at the 5'-position unmethylated cytosine bases are converted into uracil, thymidine or another base which is unlike the cytosine in terms of hybridization behavior.
  • the amplification is carried out by means of two oligonucleotides or two classes of oligonucleotides, of which one or the one class contains the four to sixteen base long sequence which is complementary to or corresponds to such a DNA as it arises would, if an equally long DNA fragment, which can bring about the specific localization of genome / chromatin sections within the cell nucleus via its sequence or secondary structure, were treated in such a way that unmethylated cytosine bases in the 5'-position in uracil, thymidine or another of the Hybridization behavior from the base which is dissimilar to the cytosine can be transformed.
  • the amplification is carried out by means of two oligonucleotides or two classes of oligonucleotides, one or one of which is one of the sequences
  • TCGCGTGTA TACACGCGA
  • TGTACGCGA TGTACGCGA
  • TCGCGTACA TTGCGTGTT, AACACGCAA, GGTACGTAA, TTACGTACC, TCGCGTGTT, AACACGCGA, GGTACGCGA, TCGCGTACG, TTGTACGCTA, TCGCGTACG
  • ATTGCGTGT ACACGCAAT, GTACGTAAT, ATTACGTAC, ATTGCGTGA, TCACGCAAT, TTACGTAAT, ATTACGTAA, ATCGCGTGA, TCACGCGAT, TTACGCGAT, ATCGCGATA, ATCGCGCTGAT, GTACTGGT
  • TGAGTTAG CTAACTCA, TTGATTTA, TAAATCAA, TGATTTAG, CTAAATCA, TTGAGTTA, TAACTCAA, TTTGGT, ACCAAA, ATTAAA, TTTAAT, TGTGGA, TCCACA, TTTATA, TATAAA, TTTGGA, TCCAAA, TTTAAA, TTTAAA, TGTGGT, ACCACA, ATTATA, TATAAT,
  • ATTAT ATAAT, GTAAT, ATTAC, ATTGT, ACAAT, GTAAT, ATTAC,
  • GAAAG CTTTC, TTTTT, AAAAA,
  • GTAAG GTAAG, CTTAC, TTTGT, ACAAA,
  • ATCGATTA ATCGATTA
  • TAATCGAT ATCGATCGAT
  • TAATCGAT ATCGATTA
  • ATCGATCGG CCGATCGAT, TCGATCGAT, ATCGATCGA, ATCGATCGT, ACGATCGAT, GCGATCGAT, ATCGATCGC,
  • TATCGATA TATCGATA, TATCGGTG, CACCGATA, TATTAATA, TATTAATA, TATTGGTG, CACCAATA,
  • TAGGTGTTA TAACACCTA, TAATATTTG, CAAATATTA, TAGGTGTTT, AAACACCTA, GAATATTTG, CAAATATTC,
  • GTAGGTGG CCACCTAC, TTATTTGT, ACAAATAA, GTAGGTGT, ACACCTAC, ATATTTGT, ACAAATAT,
  • TGCGTGGGCGG CCGCCCACGCA, TCGTTTACGTA, TACGTAAACGA, TGCGTGGGCGT, ACGCCCACGCA, ACGTTTACGTA, TACGTAAACGT,
  • TGCGTAGGCGT ACGCCTACGCA, ACGTTTACGTA, TACGTAAACGT, TGCGTAGGCGG, CCGCCTACGCA, TCGTTTACGTA, TACGTAAACGA, ATAGGAAGT, ACTTCCTAT, ATTTTTTGT, ACAAAAAAT TCGGAAGT, ACTTCCGA, ATTTTCGG, CCGAAAAT, TCGGAAGT, ACTTCCGA, GTTTTCGG, CCGAAAAC, TCGGAAAT, ATTTCCGA, ATTTTCGG, CCGAAAAT, TCGGAAAT, ATTTCCGA, GTTTTCGG, CCGAAAACGTTAATTAAT
  • TAGATAA TTATCTA, TTATTTG, CAAATAA, TTGATAA, TTATCAA, TTATTAG, CTAATAA, GATAA, TTATC, TTATT, AATAA,
  • GATAG GATAG
  • CTATC TTATT
  • AATAA AATAA
  • GATAAG AATAAG
  • CTTATC TTTATT
  • AATAAA AATAAA
  • TGTTTATTTA TAAATAAACA, TAAATAAATA, TATTTATTTA, TGTTTGTTTA, TAAACAAACA, TAAATAAATA, TATTTATTTA, TATTTATTTA, TAAATAAATA, TAAATAAATA, TATTTATTTA, TATTTGTTTA, TAAACAAATA, TAAATAATA
  • TAAAGTTTA TAAACTTTA, TGAATTTTG, CAAAATTCA, TAAAGGTTA, TAACCTTTA, TGATTTTTG, CAAAAATCA,
  • AAAGTGAAATT AATTTCACTTT, GGTTTTATTTT, AAAATAAAACC, AAAGCGAAATT, AATTTCGCTTT, GGTTTCGTTTT, AAAACGAAACC,
  • CAATTTCTCTTTTCC TAGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
  • TTATTAAAAATAGAAA TTTCTATTTTTAATAACA, TTTTTATTTTTAGTAATA, TATTACTAAAAATAAAAA, TGTTATTAAAAATAGAAT, ATTCTATTTTTAATAACA, GTTTTATTTTTAGTAATA, TATTACTAAAAATAAAAC, TTACTACAAA, TTACTAGCACCA
  • TAGGGG CCCCTA
  • TTTTTA TAAAAA
  • GAGGGG CCCCTC
  • TTTTTT AAAAAA
  • TGTTGAGTTAT ATAACTCAACA, ATGATTTAGTA, TACTAAATCAT, TGTTGATTTAT, ATAAATCAACA, GTGAGTTAGTA, TACTAACTCAC, TGTTGAGTTAT, ATAACTCAACA, ATGATTTAGTA, TACTAAATCAT, TGTTGATTTAT, ATAAATCAACA, GTGAGTTAGTA, TACTAACTCAC, GGGGATTTTT, AAAAATCCCC, GGGAATTTTT, AAAAATTCCC, GGGGATTTTT, AAAAATCCCC, GGGGATTTTT, AAAAATCCCC, GGAAATTTTT, AAAAATTTCC, GGGAATTTTT, AAAAATTCCC, GGAAATTTTT, AAAAATTTCC, GGGAATTTTT, AAAAATTCCC, GGAAATTTTT, AAAAATTTCC, GGGAATTTTT, AAAAATTCCC, GGAAATTTTT, AAAAATTTCC, GGGAATT
  • ATAAT ATAAT
  • ATTAT ATTAT
  • ATAAT GTAAT
  • ATTAC ATTAT
  • ATAAT ATAAT
  • CGTTACGGTT AACCGTAACG, AATCGTGACG, CGTCACGATT, CGTTACGGTT, AACCGTAACG, GATCGTGACG, CGTCACGATC, CGTTACGTTT, AAACGTAACG, AAGCGTGACG, CGTCACGACGTTCGGACGTC
  • TTTACGTATGA TCATACGTAAA, TTATGCGTGAA, TTCACGCATAA, TTTACGTTTGA, TCAAACGTAAA, TTAAGCGTGAA, TTCACGCTTAA, TTTACGTTTTA, TAAAACGTAAA, TGAAGCGTGAA, TTCACGGGTACA
  • TAGGTTA TAACCTA, TGATTTA, TAAATCA
  • TTTTAAATTATTTT AAAATAATTTAAAA, GGGGTGGTTTGGGG,
  • TTTTAAATTTTTTTTT AAAAAAATTTAAAA, GGGGGGGTTTGGGG,
  • TTTTAAATAATTTT AAAATTATTTAAAA
  • GGGGTTGTTTGGGG GGGGTTGTTTGGGG
  • GAGGCGGGG CCCCGCCTC, TTTCGTTTT, AAAACGAAA, GAGGTAGGG, CCCTACCTC, TTTTGTTTT, AAAACAAAA, AAGGCGGGG, CCCCGCCTT, TTTCGTTTT, AAAACGAAA, AAGGTAGGG, CCCTACCTT, TTTTGTTTT, AAAACAAAA,
  • GGGGGCGGGGT ACCCCGCCCCC, ATTTCGTTTTT, AAAAACGAAAT, GGGGGCGGGGT, ACCCCGCCCCC, GTTTCGTTTTT, AAAAACGAAAC, TATTATTTTAT, ATAAAATAATA, GTGGGGTGATAT, TATCAAGATAT, TATCAAGATAT, TATCAAGATAT, TATCAAGATAT
  • ATTACGTGAT ATCACGTAAT, ATTACGTGAT, ATCACGTAAT, ATTACGTGAT, ATCACGTAAT, GTTACGTGAT, ATCACGTAAC,
  • TTTTATATGG CCATATAAAA, TTATATAAGG, CCTTATATAA, TTATATATGG, CCATATATAA, TTATATATGG, CCATATATAA, AAATAAT, ATTATTT, GTTGTTT, AAACAAC, AAATTAA, TTAATTT, TTAGTTT, AAACTAATTATT, AAACTAATATT, AAAT
  • GGGGGTTGACGTA TACGTCAACCCCC
  • TACGTTAATTTTT AAAAATTAACGTA
  • TAACGCATACCCCC ATGATTTAGTA
  • TACTAAATCAT TGTTGAGTTAT
  • ATAACTCAACA GTTAT
  • GTTAT ATAAC
  • ATGAT ATCAT
  • TTACGTGA TCACGTAA, TTACGTGG, CCACGTAA, TTACGTGG, CCACGTAA, TTACGTGG, CCACGTAA, TTACGTGG, CCACGTAA, TTACGTGA, TCACGTAA, TTACGTGA, TCACGTAA, TTACTTA, GACGTACA, TCACGTACA
  • TGACGTGT ACACGTCA
  • ATACGTTA TAACGTAT
  • TGACGTGG CCACGTCA
  • TTACGTTA TAACGTAA
  • CGGTTATTTTG CAAAATAACCG, TAAGATGGTCG or CGACCATCTTA
  • the oligonucleotides used for the amplification contain, in addition to the consensus sequences defined above, several positions at which either one of the three bases G, A and T or any of the bases C, A and T can be present.
  • the oligonucleotides used for the amplification contain, apart from one of the consensus sequences described above, a maximum of as many additional bases as are required for the simultaneous amplification of more than one hundred different fragments per reaction from the DNA treated chemically as above.
  • a third method step the sequence context of all or part of the CpG dinucleotides or CpNpG trinucleotides contained in the amplified fragments is examined.
  • the analysis is carried out by hybridizing the fragments already provided with a fluorescence marker in the amplification to an oligonucleotide array (DNA chip).
  • the fluorescent marker can be introduced either via the primers used or through a fluorescence-labeled nucleotide (eg Cy5-dCTP, commercially available from Amersham-Pharmacia).
  • Complementary fragments hybridize to the respective oligomers immobilized on the chip surface, non-complementary fragments are removed in one or more washing steps.
  • the fluorescence at the respective hybridization sites on the chip then allows conclusions to be drawn about the sequence context of the CpG dinucleotides or CpNpG trinucleotides contained in the amplified fragments.
  • the amplified fragments are immobilized on a surface and then hybridization is carried out with a combinatorial library of distinguishable oligonucleotide or PNA oligomer probes. Again, non-complementary probes are removed by one or more washing steps.
  • the hybridized probes are either detected via their fluorescent markers or, in a further particularly preferred variant of the method, are detected using matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS) on the basis of their unique mass.
  • MALDI-MS matrix-assisted laser desorption / ionization mass spectrometry
  • the amplification products can also be influenced in terms of their average size by changing the chain extension times in the amplification step. Since mainly smaller fragments (approx. 200-500 base pairs) are examined here, a shortening of the chain extension steps is e.g. B. a PCR useful.
  • the amplified products are Gel electrophoresis is separated and the fragments in the desired size range are cut out before your analysis.
  • the amplificates cut out of the gel are amplified again using the same set of primers. Then only fragments of the desired size can be created, since others are no longer available as templates.
  • Another object of the present invention is a kit containing at least two primer pairs, reagents and auxiliary substances for amplification and / or reagents and auxiliary substances for chemical treatment and / or a combinatorial probe library and / or an oligonucleotide array (DNA chip), insofar as they are necessary or useful for carrying out the method according to the invention.
  • the CG-rich regions in the human genome are so-called CpG islands, which have a regulatory function.
  • CpG Islands in such a way that they have at least 500 bp and a GC content of> 50%, and the ratio CG / GC is> 0.6.
  • 16 Mb are CpG Islands. This means that about 0.5% of the genome sequence lies in these CpG islands, if one also considers a region up to 1000 bp downstream. This consideration is based on data from the Ensembl Database from 10/31/00, source Sanger Center. The sequence available there was approx. 3.5 GB, and the repeats were masked for the calculations.
  • the primers are AGTAGTAGTAGT (Seq. ID 1) AAAACAAAAACC (Seq. ID 2) and alternatively AGTAGTAGTAGT (Seq. ID 19) and ACAAAAACTAAA (seq. ID 20).
  • the first pair of primers leads at least to the amplificates Seq. ID 3 to 18, the second pair of primers for the amplicons of Seq. ID 21 to 31.
  • F specifies the number of predicted amplicons that can be expected if one considers N bases as a database from the genome.
  • P is the respective probability of hybridization of a primer oligonucleotide, separated after hybridization in the sense and antisense strand.
  • M is the maximum permissible length of the amplicons to be expected.
  • the probability P is determined by a first order Markov chain.
  • the assumption is made that the DNA is a random sequence depending on neighboring bases.
  • the transition probabilities of neighboring bases are necessary for the calculation of a Markov chain. These were determined empirically from 12% of the assembled human genome, which was completely treated with bisulfite, and summarized in Table 1.
  • Table 2 shows the transition probabilities for the corresponding complementary reverse Strand specified. These result from simply swapping the entries in Table 1.
  • AACAAAAACTAA 900.17 on the complementary reverse strand.
  • the primers cannot hybridize on the other strands, since no bis occur in the sense strand due to the bisulfite treatment outside the context CG and accordingly complementarily on the antisense strand.
  • An amplificate is created if, when there is a perfect base pairing on the sense strand within the maximum fragment length M, a primer on the opposite strand forms a perfect base pairing, which is the probability

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Abstract

L'invention concerne un procédé de détection parallèle de l'état de méthylation d'ADN génomique, comprenant les étapes suivantes : (a) dans un échantillon d'ADN génomique, on convertit en position 5', par traitement chimique, des bases cytosine non méthylées en uracile, thymidine ou une autre base dont le comportement d'hybridation est différent de celui de la cytosine ; b) on amplifie, dans cet ADN génomique traité chimiquement, plus de dix fragments différents présentant une longueur inférieure à 2000 paires de bases, en utilisant simultanément des oligonucléotides de synthèse comme amorces, ces dernières contenant chacune des séquences génomiques participant à au contrôle de l'expression génique et/ou transcrites et/ou traduites, similaires à celles qui seraient obtenues après le traitement de l'étape a) ; c) on détermine le contexte des séquences de la totalité ou d'une partie des CpG dinucléotides ou trinucléotides CpNpG contenus dans les fragments amplifiés.
EP00989842A 1999-12-06 2000-12-06 Procede de detection parallele de l'etat de methylation d'adn genomique Withdrawn EP1238112A2 (fr)

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DE19959691 1999-12-06
DE19959691A DE19959691A1 (de) 1999-12-06 1999-12-06 Verfahren zur parallelen Detektions des Methylierungszustandes von genomischer DNA
PCT/DE2000/004381 WO2001042493A2 (fr) 1999-12-06 2000-12-06 Procede de detection parallele de l'etat de methylation d'adn genomique

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AU2663201A (en) 2001-06-18
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