Method and devices for dna methylation analysis
First Claim
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1. A method for the amplification of genomic DNA whereby the cytosine methylation pattern of the genomic DNA is retained in the amplificate sequence(s), said method comprising the following steps:
- (a) heating the genomic DNA to a temperature operative to cause denaturation;
(b) cooling the denatured DNA in the presence of single stranded oligonucleotide primers such that the primers anneal to the DNA;
(c) heating the mixture in the presence of a polymerase and nucleotides to a temperature such that the primers are extended;
(d) contacting the double stranded nucleic acid with a methyltransferase and a methyl donor molecule under conditions conducive to the methylation of the synthesised strand such that the CpG dinucleotides within the synthesised strand are methylated according to the methylation status of the corresponding CpG dinucleotide on the template strand thereby preserving the genomic methylation pattern;
(e) repeating steps A-D a desired number of times to reach a desired number of nucleic acids.
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Abstract
The invention outlines a method for the methylation pattern retaining amplification of nucleic acid molecules. Furthermore the invention describes several devices for use in the methylation pattern retaining amplification of nucleic acid molecules.
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Citations
15 Claims
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1. A method for the amplification of genomic DNA whereby the cytosine methylation pattern of the genomic DNA is retained in the amplificate sequence(s), said method comprising the following steps:
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(a) heating the genomic DNA to a temperature operative to cause denaturation;
(b) cooling the denatured DNA in the presence of single stranded oligonucleotide primers such that the primers anneal to the DNA;
(c) heating the mixture in the presence of a polymerase and nucleotides to a temperature such that the primers are extended;
(d) contacting the double stranded nucleic acid with a methyltransferase and a methyl donor molecule under conditions conducive to the methylation of the synthesised strand such that the CpG dinucleotides within the synthesised strand are methylated according to the methylation status of the corresponding CpG dinucleotide on the template strand thereby preserving the genomic methylation pattern;
(e) repeating steps A-D a desired number of times to reach a desired number of nucleic acids. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
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Specification