Ligase/polymerase 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 two oligonucleotides, the latter being joined by inserting at least one oligonucleotide. In the ligation product, one nucleotide carries a detectable label, and the elongation depends 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.
Claims38
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.
A method for detecting 5-methylcytosine in at least one genomic DNA sample, said method comprising the steps of:
- (a) chemically converting genomic DNA from at least one DNA sample with a reagent, whereby 5-methylcytosine and cytosine react differently, thus exhibiting different base pairing behaviors in the DNA duplex subsequent to the reaction;
- (b) then, amplifying the pretreated DNA using a polymerase and at least one oligonucleotide (type A) as a primer;
- (c) then, hybridizing at least one set of oligonucleotides to the amplified genomic DNA, forming a duplex, one set of oligonucleotides comprising different species of type B and of type C, 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, 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, elongating the at least one oligonucleotide (type B) having a known sequence 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 5′-end of the oligonucleotide of type C, the elongation depending on the methylation status of the specific cyto sine in the genomic DNA sample;
- (e) then, incubating the oligonucleotides 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 ligated to produce 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, detecting whether a ligation product has formed for each analyzed position.
Dependencies are inferred from the claim text.