Method for detecting methylated cytosine by using bisulfite reaction
View Patent ↗The invention provides a method for converting non-methylated cytosine in a single-stranded DNA into uracil by a bisulfite reaction with a high conversion efficiency from non-methylated cytosine into uracil. The invention also provides a method for amplifying the single-stranded DNA in which non-methylated cytosine has been converted into uracil, as well as a method for detecting methylated cytosine in the single-stranded DNA.
1. A method for converting non-methylated cytosine in a single-stranded DNA to uracil, comprising subjecting the single-stranded DNA to a bisulfite reaction in the presence of at least one compound selected from the group consisting of:
a compound of formula [1]
wherein R 1 to R 6 represent each independently a hydrogen atom or an alkyl group having 1 to 3 carbon atoms; and n represents an integer of 1 to 3; and
a compound of formula [2]
wherein Y represents a carbon atom; R 7 to R 12 represent each independently a hydrogen atom or an alkyl group having 1 to 3 carbon atoms; and k represents an integer of 2.
2. The method according to claim 1 , wherein R 1 to R 6 are all hydrogen atoms.
3. The method according to claim 1 , wherein R 7 to R 11 are all hydrogen atoms, R 12 is an alkyl group having 1 to 3 carbon atoms, and k is an integer of 2.
4. The method according to claim 1 , comprising subjecting a single-stranded DNA to a bisulfite reaction in the presence of at least one compound of formula [1].
5. A method for amplifying single-stranded DNA in which non-methylated cytosine has been converted into uracil, comprising providing a single-stranded DNA in which non-methylated cytosine has been converted into uracil in accordance with the method of claim 1 , and subjecting the single-stranded DNA in which non-methylated cytosine has been converted into uracil to a PCR reaction.
6. A method for detecting methylated cytosine in a single-stranded DNA, comprising proving an amplified single-stranded DNA in which non-methylated cytosine has been converted into uracil in accordance with claim 5 , and subjecting the amplified single-stranded DNA to nucleotide sequence analysis to detect methylated cytosine therein.
7. The method according to claim 4 , wherein R 1 to R 6 are all hydrogen atoms.
8. The method according to claim 1 , comprising subjecting a single-stranded DNA to a bisulfite reaction in the presence of at least one compound of formula [2].
9. The method according to claim 8 , wherein R 7 to R 11 are all hydrogen atoms, R 12 is an alkyl group having 1 to 3 carbon atoms, and k is an integer of 2.
10. A method for amplifying single-stranded DNA in which non-methylated cytosine has been converted into uracil, comprising providing a single-stranded DNA in which non-methylated cytosine has been converted into uracil in accordance with the method of claim 4 , and subjecting the single-stranded DNA in which non-methylated cytosine has been converted into uracil to a PCR reaction.
11. A method for detecting methylated cytosine in a single-stranded DNA, comprising providing an amplified single-stranded DNA in which non-methylated cytosine has been converted into uracil in accordance with claim 10 , and subjecting the amplified single-stranded DNA to nucleotide sequence analysis to detect methylated cytosine therein.
12. A method for amplifying single-stranded DNA in which non-methylated cytosine has been converted into uracil, comprising providing a single-stranded DNA in which non-methylated cytosine has been converted into uracil in accordance with the method of claim 7 , and subjecting the single-stranded DNA in which non-methylated cytosine has been converted into uracil to a PCR reaction.
13. A method for detecting methylated cytosine in a single-stranded DNA, comprising providing an amplified single-stranded DNA in which non-methylated cytosine has been converted into uracil in accordance with claim 12 , and subjecting the amplified single-stranded DNA to nucleotide sequence analysis to detect methylated cytosine therein.
14. A method for amplifying single-stranded DNA in which non-methylated cytosine has been converted into uracil, comprising providing a single-stranded DNA in which non-methylated cytosine has been converted into uracil in accordance with the method of claim 8 , and subjecting the single-stranded DNA in which non-methylated cytosine has been converted into uracil to a PCR reaction.
15. A method for detecting methylated cytosine in a single-stranded DNA, comprising providing an amplified single-stranded DNA in which non-methylated cytosine has been converted into uracil in accordance with claim 14 , and subjecting the amplified single-stranded DNA to nucleotide sequence analysis to detect methylated cytosine therein.
16. A method for amplifying single-stranded DNA in which non-methylated cytosine has been converted into uracil, comprising providing a single-stranded DNA in which non-methylated cytosine has been converted into uracil in accordance with the method of claim 9 , and subjecting the single-stranded DNA in which non-methylated cytosine has been converted into uracil to a PCR reaction.
17. A method for detecting methylated cytosine in a single-stranded DNA, comprising providing an amplified single-stranded DNA in which non-methylated cytosine has been converted into uracil in accordance with claim 16 , and subjecting the amplified single-stranded DNA to nucleotide sequence analysis to detect methylated cytosine therein.