METHOD FOR DETECTING CYTOSINE METHYLATION IN DNA SAMPLES
First Claim
1. A method for detecting 5-methylcytosine in genomic DNA samples, comprising:
- (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 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) having a known sequence of n nucleotides, forming a duplex, said hybridized oligonucleotides of type B, with their 3′
-ends, completely hybridizing to the CpG positions to be analyzed with regard to their methylation in the genomic DNA sample;
(d) elongating the oligonucleotide (type B), provided that it has previously hybridized with its 3′
end to the position to be analyzed without mispairings, by at least one nucleotide by means of a polymerase, 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
(e) analyzing the elongated oligonucleotides for the presence of the label, wherein the presence of the label is indicative of the presence of 5-methylcytosine.
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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.
1 Citation
26 Claims
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1. A method for detecting 5-methylcytosine in genomic DNA samples, comprising:
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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 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) having a known sequence of n nucleotides, forming a duplex, said hybridized oligonucleotides of type B, with their 3′
-ends, completely hybridizing to the CpG positions to be analyzed with regard to their methylation in the genomic DNA sample;(d) elongating the oligonucleotide (type B), provided that it has previously hybridized with its 3′
end to the position to be analyzed without mispairings, by at least one nucleotide by means of a polymerase, 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(e) analyzing the elongated oligonucleotides for the presence of the label, wherein the presence of the label is indicative of the presence of 5-methylcytosine. - 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)
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Specification