发明公开
EP1366190A2 VERFAHREN ZUR QUANTIFIZIERUNG VON CYTOSIN-METHYLIERUNGEN IN KOMPLEX AMPLIFIZIERTER GENOMISCHER DNA
审中-公开
定量胞嘧啶甲基化方法在复杂基因组DNA扩增
- 专利标题: VERFAHREN ZUR QUANTIFIZIERUNG VON CYTOSIN-METHYLIERUNGEN IN KOMPLEX AMPLIFIZIERTER GENOMISCHER DNA
- 专利标题(英): Method for quantifying cytosine methylations in genomic dna that is amplified in a complex manner
- 专利标题(中): 定量胞嘧啶甲基化方法在复杂基因组DNA扩增
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申请号: EP01989384.1申请日: 2001-12-05
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公开(公告)号: EP1366190A2公开(公告)日: 2003-12-03
- 发明人: OLEK, Alexander
- 申请人: Epigenomics AG
- 申请人地址: Kastanienallee 24 10435 Berlin DE
- 专利权人: Epigenomics AG
- 当前专利权人: Epigenomics AG
- 当前专利权人地址: Kastanienallee 24 10435 Berlin DE
- 代理机构: Schubert, Klemens, Dr.
- 优先权: DE10061348 20001206
- 国际公布: WO02046452 20020613
- 主分类号: C12Q1/68
- IPC分类号: C12Q1/68
摘要:
The invention relates to a method for providing demethylated DNA as a reference material for analyzing cytosine methylations in genomic DNA samples while using complex amplifications. To this end, the following method steps are carried out: a) a genomic DNA sample is amplified with primers that are either very short or degenerated oligonucleotides or oligonucleotides that are complementary to adapters. In the second case, the DNA sample is cut by a restriction enzyme before amplification, and the adapters, understood as short nucleotide fragments of a known sequence, ligate to the ends of the resulting DNA fragments. The amplificates are chemically treated in such a manner that cytosine bases, which are unmethylated at the 5-position, are transformed into uracil, thymine or into another base that is dissimilar to cytosine with regard to hybridization behavior, whereas the 5-methylcytosine bases remain essentially unchanged. This is understood in the following as chemical pretreatment. The chemically pretreated amplificates are, in turn, amplified. To this end, a number of specifically hybridizing oligonucleotides or oligonucleotides that are complementary to the adapters are used as primers. In the latter case, the chemical pretreatment is also carried out.
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