Analysis of methylation patterns in genomic dna using bisulfite treatment and fluorescence polarisation assay techniques

摘要

A method for the analysis of the methylation of cytosine bases in genomic DNA samples, comprising the following steps: (a) the genomic DNA is chemically treated in such a manner that cytosine is converted into uracil or a similar base regarding the base pairing behaviour in the DNA duplex, 5 methylcytosine however remains unchanged; (b) the chemically treated DNA is amplified using of at least one species of oligonucleotide (type A) as a primer in a polymerase reaction; (c) the amplificate is left in solution with one or more species of fluorophore labelled nucleotides and one or more species of oligonucleotide (type B), wherein the type B oligonucleotide hybridises under appropriate conditions with its 3' end directly on or up to 10 bases from the position to be examined, and wherein said type B oligonucleotide is at least partly nuclease resistant; (d) the hybridised oligonucleotide (type B) is extended by means of a polymerase by at least one nucleotide, whereby the extension is dependent upon the methylation status of the respective cytosine position in the genomic DNA sample; (e) the solution is incubated with a phosphodiesterase, which is capable of digesting nucleic acids, however incompletely digests the type B oligonucleotides and its extension products; (f) the fluorescence polarisation of the solution is measured whereby for each fluorescent label used one determines the degree of polarisation.