| 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. |
