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US 2011/0086356 A1Published application
Method for measuring dna methylation
AssigneeSumitomo Chemical Company, Limited
InventorsYoshitaka Tomigahara, Hideo Satoh, Hirokazu Tarui
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Abstract
The present invention relates to a method of measuring the content of methylated DNA in a DNA region of interest in a genomic DNA contained in a biological specimen, and so on.
Claims26
A method of measuring the content of methylated DNA in an objective DNA region in genomic DNA contained in a biological specimen, comprising:
- (1) First step of subjecting a DNA sample derived from genomic DNA contained in a biological specimen to a digestion treatment with a methylation-sensitive restriction enzyme;
- (2) Second step of obtaining a single-stranded DNA (plus strand) containing the objective DNA region from the DNA sample that has been subjected to the digestion treatment and obtained in First step, and causing base-pairing between the single-stranded DNA (plus strand) and a single-stranded immobilized oligonucleotide comprising a nucleotide sequence complementary to a part (provided that, not containing the objective DNA region) of 3′-end of the single-stranded DNA, thereby selecting the single-stranded DNA,
- (3) Third step of extensionally-forming a double-stranded DNA from the single-stranded DNA by allowing a single extension of a primer using the single-stranded DNA selected in Second step as a template and the single-stranded immobilized oligonucleotide as the primer, and
- (4) Fourth step comprising, as a pre step of the following regular steps, a step (pre step) of temporarily separating the extensionally-formed double-stranded DNA obtained in Third step into a single-stranded state, and as regular steps:
- (a) Step A (regular step) comprising Step A1 of selecting the DNA in a single-stranded state by causing base-pairing between the generated DNA in a single-stranded state (plus strand) and the single-stranded immobilized oligonucleotide (minus strand), and Step A2 of extensionally-forming double-stranded DNA from the DNA in a single-stranded state by causing one extension of a primer by using the DNA in a single-stranded state selected in Step A1 as a template and the single-stranded immobilized oligonucleotide as the primer, and
- (b) Step B (regular step) of extensionally-forming double-stranded DNA from the DNA in a single-stranded state by causing one extension of an extension primer by using the generated DNA in a single-stranded state (minus strand) as a template, and a primer (reverse primer) comprising a nucleotide sequence (plus strand) complementary to a partial nucleotide sequence (minus strand) of the nucleotide sequence of the DNA in a single-stranded state (minus strand), wherein the partial nucleotide sequence (minus strand) is located on further 3′-end side than the 3′-end of the nucleotide sequence (minus strand) complementary to the nucleotide sequence (plus strand) of the objective DNA region, as the extension primer,
- wherein the methylated DNA in the objective DNA region is amplified to a detectable level by repeating each regular step after temporarily separating the extensionally-formed double-stranded DNA obtained in the regular steps into a single-stranded state, and an amount of the amplified DNA is quantified.
4 more elements
A method of measuring the content of methylated DNA in an objective DNA region possessed by genomic DNA contained in a biological specimen, comprising:
- (1) First step of subjecting a DNA sample derived from genomic DNA contained in a biological specimen to a digestion treatment with a methylation sensitive restriction enzyme;
- (2) Second step of obtaining single-stranded DNA (plus strand) containing the objective DNA region from the DNA sample that has been subjected to the digestion treatment and obtained in First step, and causing base-pairing between the single-stranded DNA (plus strand) and a single-stranded immobilized oligonucleotide comprising a nucleotide sequence complementary to a part of the single-stranded DNA, thereby selecting the single-stranded DNA,
- (3) Third step comprising a step of temporarily separating the single-stranded DNA selected in Second step into a single-stranded state, and extensionally-forming double-stranded DNA from the single-stranded DNA by allowing one extension of a primer using the generated DNA in a single-stranded state (plus strand) as a template and a forward primer comprising a nucleotide sequence (minus strand) complementary to a partial nucleotide sequence (plus strand) located on further 3′-end side than the 3′-end of the nucleotide sequence (plus strand) of the objective DNA region as an extension primer,
- (4) Fourth step comprising, as a pre step of each of the following regular steps, a step (pre step) of temporarily separating the extensionally-formed double-stranded DNA obtained in Third step into a single-stranded state, and as regular steps:
- (a) Step A (Regular step) comprising Step A1 of selecting the DNA in a single-stranded state by causing base-pairing between the generated DNA in a single-stranded state (plus strand) and the forward primer (minus strand), and Step A2 of extensionally-forming double-stranded DNA from the DNA in a single-stranded state by causing one extension of a primer by using the DNA in a single-stranded state selected in Step A1 as a template and the forward primer as an extension primer, and
- (b) Step B (Regular step) of extensionally-forming double-stranded DNA from the DNA in a single-stranded state by causing one extension of an extension primer by using the generated DNA in a single-stranded state (minus strand) as a template, and a reverse primer comprising a nucleotide sequence (plus strand) complementary to a partial nucleotide sequence (minus strand) of the nucleotide sequence of the DNA in a single-stranded state (minus strand), wherein the partial nucleotide sequence (minus strand) is located on further 3′-end side than the 3′-end of the nucleotide sequence (minus strand) complementary to the nucleotide sequence (plus strand) of the objective DNA region, as the extension primer,
- wherein the methylated DNA in the objective DNA region is amplified to a detectable level by repeating each regular step in Fourth step after temporarily separating the extensionally-formed double-stranded DNA obtained in the regular steps into a single-stranded state, and an amount of the amplified DNA is quantified.
4 more elements
Dependencies are inferred from the claim text.