METHOD FOR AMPLIFYING METHYLATED DNA, METHOD FOR DETERMINING METHYLATION OF DNA, AND METHOD FOR DETERMINING CANCER
An object of the present invention is to provide a method capable of conveniently carrying out amplification of methylated DNA, and determination of methylation of DNA and determination of the occurrence of cancer using the amplification of methylated DNA. The present invention relates to “a method for amplifying a methylated analysis target region; a method for determining the methylation of an analysis target region; a reagent for use in the determination of methylation; a method for determining the occurrence of cancer; a method for obtaining data for determining the occurrence of cancer; a reagent for use in the determination of the occurrence of cancer; and a marker for use in the determination of the occurrence of cancer”.
1 . A method for amplifying a methylated analysis target region in double-stranded DNA, comprising the following steps 1 and 2:
(1) a step 1 of treating double-stranded DNA containing an analysis target region with S1 nuclease and a methylation-sensitive restriction enzyme; and
(2) a step 2 of amplifying the analysis target region of the double-stranded DNA treated in the step 1.
2 . The amplification method according to claim 1 , wherein, in the step 1, treatment with S1 nuclease is carried out and then treatment with a methylation-sensitive restriction enzyme is carried out.
3 . The amplification method according to claim 1 , wherein, in the step 1, treatment with a methylation-sensitive restriction enzyme is carried out and then treatment with S1 nuclease is carried out.
4 . The amplification method according to claim 1 , further comprising:
a fragmentation step of treating double-stranded DNA with a restriction enzyme whose recognition sequence is not present in the analysis target region, prior to the step 1.
5 . A method for determining the methylation of an analysis target region in double-stranded DNA, comprising the following steps 1 to 4:
(1) a step 1 of treating double-stranded DNA containing the analysis target region with S1 nuclease and a methylation-sensitive restriction enzyme;
(2) a step 2 of amplifying the analysis target region of the double-stranded DNA treated in the step 1;
(3) a step 3 of confirming the presence or absence of an amplification product obtained in the step 2; and
(4) a step 4 of determining whether or not the analysis target region is methylated based on the results of the step 3.
6 . The methylation determination method according to claim 5 , wherein, in the step 1, treatment with S1 nuclease is carried out and then treatment with a methylation-sensitive restriction enzyme is carried out.
7 . The methylation determination method according to claim 5 , wherein, in the step 1, treatment with a methylation-sensitive restriction enzyme is carried out and then treatment with S1 nuclease is carried out.
8 . The methylation determination method according to claim 5 , further comprising:
a fragmentation step of treating double-stranded DNA with a restriction enzyme whose recognition sequence is not present in the analysis target region, prior to the step 1.
9 . (canceled)
10 . A method for determining cancer, comprising the following steps 1 to 4:
(1) a step 1 of treating double-stranded DNA containing an analysis target region with S1 nuclease and a methylation-sensitive restriction enzyme;
(2) a step 2 of amplifying the analysis target region of the double-stranded DNA treated in the step 1;
(3) a step 3 of confirming the presence or absence of an amplification product obtained in the step 2; and
(4) a step 4 of determining cancer based on the results of the step 3.
11 . The cancer determination method according to claim 10 , wherein, in the step 1, treatment with S1 nuclease is carried out and then treatment with a methylation-sensitive restriction enzyme is carried out.
12 . The cancer determination method according to claim 10 , wherein, in the step 1, treatment with a methylation-sensitive restriction enzyme is carried out and then treatment with S1 nuclease is carried out.
13 . The cancer determination method according to claim 10 , further comprising:
a fragmentation step of treating DNA with a restriction enzyme whose recognition sequence is not present in an analysis target region, prior to the step 1.
14 . The cancer determination method according to claim 10 , wherein the analysis target region is a base sequence containing a base sequence represented by SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 11.
15 - 17 . (canceled)