Method for detecting cytosine methylation in DNA samples
First Claim
1. A method for concurrently detecting 5-methylcytosine and SNP in genomic DNA samples, characterized in that the following steps are carried out:
- (a) chemically converting a genomic DNA from a DNA sample with a reagent, 5-methylcytosine and cytosine reacting differently, thus exhibiting different base pairing behaviors in the DNA duplex subsequent to the reaction;
(b) amplifying the pretreated DNA using a polymerase and at least one oligonucleotide (type A) as a primer;
(c) hybridizing the amplified genomic DNA to at least one oligonucleotide (type B), forming a duplex, 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;
(d) elongating the oligonucleotide (type B) having a known sequence of n nucleotides by means of a polymerase by a plurality of nucleotides, at least one nucleotide carrying a detectable label, and the elongation depending on the methylation status of the specific cytosine in the genomic DNA sample and on a single nucleotide polymorphism;
(e) analyzing the elongated oligonucleotides for the presence of the label; and
(f) deducing the presence of said 5-methylcytosine and SNP in the genomic DNA samples from the incorporated label.
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Abstract
Described is a method for detecting 5-methylcytosine in genomic DNA samples. First, a genomic DNA from a DNA sample is chemically converted with a reagent, 5-methylcytosine and cytosine reacting differently, and the pretreated DNA is subsequently amplified using a polymerase and at least one primer. In the next step, the amplified genomic DNA is hybridized to at least one oligonucleotide, forming a duplex, and said oligonucleotide is elongated by at least one nucleotide, the nucleotide carrying a detectable label, and the elongation depending on the methylation status of the specific cytosine in the genomic DNA sample. In the next step, the elongated oligonucleotides are analyzed for the presence of the label.
43 Citations
29 Claims
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1. A method for concurrently detecting 5-methylcytosine and SNP in genomic DNA samples, characterized in that the following steps are carried out:
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(a) chemically converting a genomic DNA from a DNA sample with a reagent, 5-methylcytosine and cytosine reacting differently, thus exhibiting different base pairing behaviors in the DNA duplex subsequent to the reaction; (b) amplifying the pretreated DNA using a polymerase and at least one oligonucleotide (type A) as a primer; (c) hybridizing the amplified genomic DNA to at least one oligonucleotide (type B), forming a duplex, 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;(d) elongating the oligonucleotide (type B) having a known sequence of n nucleotides by means of a polymerase by a plurality of nucleotides, at least one nucleotide carrying a detectable label, and the elongation depending on the methylation status of the specific cytosine in the genomic DNA sample and on a single nucleotide polymorphism; (e) analyzing the elongated oligonucleotides for the presence of the label; and (f) deducing the presence of said 5-methylcytosine and SNP in the genomic DNA samples from the incorporated label. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29)
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Specification