Method for detecting cytosine methylation in dna samples
AssigneeEpigenomics AG
InventorsAlexander Olek, Kurt Berlin
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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.
Claims26
A method for detecting 5-methylcytosine in genomic DNA samples, characterized in that the following steps are carried out:
- (a) a genomic DNA from a 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) pretreated DNA is amplified using a polymerase and at least one oligonucleotide (type A) as a primer;
- (c) the amplified genomic DNA is hybridized 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, partially or completely hybridizing to the positions to be analyzed with regard to their methylation in the genomic DNA sample;
- (d) the oligonucleotide (type B), provided that it has previously hybridized with its 3′-terminus to the position to be analyzed without mispairings, is elongated 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;
- (e) the elongated oligonucleotides are analyzed for the presence of the label.
The method as recited in one of the preceding claims, characterized in that, prior to the amplification, the DNA is treated with a bisulfite solution (=disulfite, hydrogen sulfite).
The method as recited in one of the preceding claims, characterized in that the amplification is carried out by means of the polymerase chain reaction (PCR).
The method as recited in one of the preceding claims, characterized in that the labels of the nucleotides are fluorescence labels.
The method as recited in one of the preceding claims, characterized in that the detection of the elongated oligonucleotides is carried out and visualized by means of matrix assisted laser desorption/ionization mass spectrometry (MALDI) or using electron spray mass spectrometry (ESI).
The method as recited in one of the preceding claims, wherein the polymerases are heat-resistant DNA-polymerases.
The method as recited in one of the preceding claims, wherein SNPs are also detected and visualized in addition to the DNA methylation.
The method as recited in one of the preceding claims, wherein the used nucleotides are terminating (type C 2) and/or chain-elongating nucleotides (type C 1).
The method as recited in one of the preceding claims, wherein the nucleotides (type C 1 and C 2) are selected from a group comprising either the nucleobases A, T and C or else the bases G and A and T.
The method as recited in one of the preceding claims, characterized in that the amplification of several DNA segments is carried out in one reaction vessel.
The method as recited in one of the preceding claims, characterized in that the methylation analyses of the upper and lower DNA strands are carried out in one experiment.
Dependencies are inferred from the claim text.