Patent ID: 7405040
Filing Date: 2008-07-29
Classification: C12Q

Abstract:
1. A method for detecting 5-methylcytosine in at least one genomic DNA sample, characterized in that the following steps are carried out: (a) genomic DNA from at least one DNA sample is chemically converted with a reagent, 5-methylcytosine and cytosine reacting differently, thus exhibiting different base pairing behaviors in the DNA duplex subsequent to the reaction; (b) then, the pretreated DNA is amplified using a polymerase and at least one oligonucleotide (type A) as a primer; (c) then, at least one set of oligonucleotides is hybridized to the amplified genomic DNA, forming a duplex; one set of oligonucleotides comprising different species of type B and of type C; and said hybridized oligonucleotides of type B, with their 3′-ends, immediately or at a distance of up to 10 bases, adjoining the positions to be analyzed with regard to their methylation in the genomic DNA sample; and said second oligonucleotide (type C) hybridizing to a second region of the target molecule so that the 5′-end of the second oligonucleotide (type C) is separated from the 3′-end of the first oligonucleotide (type B) at the location of said selected position by a gap of the size of a single nucleotide or of up to 10 nucleotides; (d) then, the at least one oligonucleotide (type B) having a known sequence is elongated by means of a polymerase by not more than the number of nucleotides lying between the 3′-end of the oligonucleotide of type B and the 5t-end of the oligonucleotide of type C, the elongation depending on the methylation status of the specific cytosine in the genomic DNA sample; (e) then, the oligonucleotides are incubated in the presence of a ligase, the adjoining first oligonucleotide of type B elongated by the polymerase reaction and the second oligonucleotide of type C being linked, resulting in a ligation product provided that the oligonucleotide of type B has been elongated in the preceding step in such a manner that now the 3′-end with existing 3′-hydroxy function immediately adjoins the 5′-end of the oligonucleotide of type C; (f) then, it is detected whether a ligation product has formed for each analyzed position.