BNA clamp method
The invention provides a method of selectively amplifying a detection target nucleic acid by inhibiting amplification of a detection non-target nucleic acid (e.g., wild-type gene) in a test sample by using, in a nucleic acid amplification reaction, an oligonucleotide analog containing one or more kinds of one or more unit structures of various nucleoside analogs represented by the following formula (I): wherein the symbols are as defined in the DESCRIPTION, and the like, or a salt thereof, as a clamp nucleic acid, and detecting the amplified nucleic acid.
1. A method of detecting a target nucleic acid having a difference in a base sequence in a detection target site in the target nucleic acid in a test sample, wherein the target nucleic acid comprises at least one difference in the base sequence from a detection non-target nucleic acid, comprising
a step of selectively amplifying a region containing at least a part of the detection target site of the detection target nucleic acid in the test sample by a nucleic acid amplification method using a clamp nucleic acid having a base sequence complementary to the base sequence of the detection target site in the detection non-target nucleic acid, and
a step of detecting the amplified nucleic acid,
wherein the clamp nucleic acid is an oligonucleotide analog containing one or more of a unit structure of nucleoside analog represented by the following formula (I):
wherein
Base is a pyrimidine or purine nucleic acid base optionally having one or more substituents,
R 1 is a hydrogen atom, an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a sulfonyl group, a silyl group, or a functional molecule unit substituent,
m is an integer of 0-2, and
n1 is an integer of 1-3,
provided a binding form between respective nucleoside analogs in the oligonucleotide analogs optionally contains one or more phosphorothioate bonds in addition to a phosphodiester bond and, when two or more of the aforementioned unit structures are contained, Base may be the same or different between the structures, or a salt thereof.
2. The method according to claim 1 , wherein the clamp nucleic acid is an oligonucleotide analog containing one or more of the unit structure of nucleoside analog of the above-mentioned formula (I), wherein
Base is a pyrimidine or purine nucleic acid base optionally having one or more substituents selected from the group consisting of a hydroxyl group, a hydroxyl group protected with a protective group for nucleic acid synthesis, an alkoxy group having 1 to 5 carbon atoms, a mercapto group, a mercapto group protected with a protective group for nucleic acid synthesis, an alkylthio group having 1 to 5 carbon atoms, an amino group, an amino group protected with a protective group for nucleic acid synthesis, an amino group substituted by an alkyl group having 1 to 5 carbon atoms, an alkyl group having 1 to 5 carbon atoms, and a halogen atom,
R 1 is a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, a benzyl group, an acetyl group, a benzoyl group, a methanesulfonyl group, or a p-toluenesulfonyl group,
m is an integer of 0-2, and
n1 is an integer of 1-3,
or a salt thereof.
3. The method according to claim 1 , wherein the clamp nucleic acid is an oligonucleotide analog containing one or more of the unit structure of nucleoside analog of the above-mentioned formula (I), wherein
Base is a pyrimidine or purine nucleic acid base optionally having one or more substituents selected from the group consisting of a hydroxyl group, a hydroxyl group protected with a protective group for nucleic acid synthesis, an alkoxy group having 1 to 5 carbon atoms, a mercapto group, a mercapto group protected with a protective group for nucleic acid synthesis, an alkylthio group having 1 to 5 carbon atoms, an amino group, an amino group protected with a protective group for nucleic acid synthesis, an amino group substituted by an alkyl group having 1 to 5 carbon atoms, an alkyl group having 1 to 5 carbon atoms, and a halogen atom,
R 1 is a methyl group,
m is 0, and
n1 is 1,
or a salt thereof.
4. The method according to claim 1 , wherein the clamp nucleic acid has a length of 5-30 mer.
5. The method according to claim 1 , wherein the nucleic acid amplification method is a polymerase chain reaction (PCR) method.
6. The method according to claim 5 , wherein the PCR method is a real-time PCR method.
7. The method according to claim 6 , wherein the real-time PCR method is performed using a detection probe, and the detection probe is a single strand nucleic acid having a base sequence complementary to the base sequence of the region to be detected for a difference in a base sequence in a detection target site, wherein one terminal is substituted by a fluorescence group, the other terminal is substituted by a quenching group.
8. The method according to claim 7 , wherein the detection probe is an oligonucleotide analog containing one or more of the unit structure of nucleoside analog of the above-mentioned formula (I), or a salt thereof.
9. The method according to claim 1 , wherein the difference in the base sequence between the detection non-target nucleic acid and the detection target nucleic acid is caused by one or more mutations selected from the group consisting of substitution, insertion, deletion, inversion, overlap and translocation or a combination thereof.
10. The method according to claim 1 , wherein the step of detecting the amplified nucleic acid comprises sequencing of an amplification product.
11. The method according to claim 1 , wherein the target nucleic acid is a gene, and the difference in the base sequence of the gene to be the detection target relates to the onset and/or treatment sensitivity of a particular disease.
12. A kit for detection of a target nucleic acid having a difference in a base sequence in a detection target site in the target nucleic acid in a test sample, wherein the target nucleic acid is a detection target nucleic acid having at least one difference in a base sequence from a detection non-target nucleic acid, the kit comprising
(a) a clamp nucleic acid having a base sequence complementary to the base sequence of the detection target site of a detection non-target nucleic acid, and
(b) a reagent for selectively amplifying a region containing at least a part of the detection target site of the detection target nucleic acid in the test sample,
wherein the clamp nucleic acid is an oligonucleotide analog containing one or more of a unit structure of nucleoside analog represented by the following formula (I):
wherein
Base is a pyrimidine or purine nucleic acid base optionally having one or more substituents,
R 1 is a hydrogen atom, an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a sulfonyl group, a silyl group, or a functional molecule unit substituent,
m is an integer of 0-2, and
n1 is an integer of 1-3,
provided a binding form between respective nucleoside analogs in the oligonucleotide analogs optionally contains one or more phosphorothioate bonds in addition to a phosphodiester bond and, when two or more of the aforementioned unit structures are contained, Base may be the same or different between the structures, or a salt thereof.
13. The kit according to claim 12 , wherein the clamp nucleic acid is an oligonucleotide analog containing one or more of the unit structure of nucleoside analog of the above-mentioned formula (I), wherein
Base is a pyrimidine or purine nucleic acid base optionally having one or more substituents selected from the group consisting of a hydroxyl group, a hydroxyl group protected with a protective group for nucleic acid synthesis, an alkoxy group having 1 to 5 carbon atoms, a mercapto group, a mercapto group protected with a protective group for nucleic acid synthesis, an alkylthio group having 1 to 5 carbon atoms, an amino group, an amino group protected with a protective group for nucleic acid synthesis, an amino group substituted by an alkyl group having 1 to 5 carbon atoms, an alkyl group having 1 to 5 carbon atoms, and a halogen atom,
R 1 is a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, a benzyl group, an acetyl group, a benzoyl group, a methanesulfonyl group, or a p-toluenesulfonyl group,
m is an integer of 0-2, and
n1 is an integer of 1-3,
or a salt thereof.
14. The kit according to claim 12 , wherein the clamp nucleic acid is an oligonucleotide analog containing one or more of the unit structure of nucleoside analog of the above-mentioned formula (I), wherein
Base is a pyrimidine or purine nucleic acid base optionally having one or more substituents selected from the group consisting of a hydroxyl group, a hydroxyl group protected with a protective group for nucleic acid synthesis, an alkoxy group having 1 to 5 carbon atoms, a mercapto group, a mercapto group protected with a protective group for nucleic acid synthesis, an alkylthio group having 1 to 5 carbon atoms, an amino group, an amino group protected with a protective group for nucleic acid synthesis, an amino group substituted by an alkyl group having 1 to 5 carbon atoms, an alkyl group having 1 to 5 carbon atoms, and a halogen atom,
R 1 is a methyl group,
m is 0, and
n1 is 1,
or a salt thereof.
15. The kit according to claim 12 , wherein the clamp nucleic acid has a length of 5-30 mer.
16. The kit according to claim 12 , wherein the reagent of the aforementioned (b) comprises an amplification primer for polymerase chain reaction (PCR).
17. The kit according to claim 16 , wherein the reagent of the aforementioned (b) further comprises a reagent for real-time PCR.
18. The kit according to claim 17 , wherein the real-time PCR method is performed using a detection probe, and the detection probe is a single strand nucleic acid having a base sequence complementary to the base sequence of the region to be detected for a difference in a base sequence in a detection target nucleic acid, wherein one terminal is substituted by a fluorescence group, the other terminal is substituted by a quenching group.
19. The kit according to claim 18 , wherein the detection probe is an oligonucleotide analog containing one or more of the unit structure of nucleoside analog of the above-mentioned formula (I), or a salt thereof.
20. The kit according to claim 12 , wherein the difference in the base sequence between the detection non-target nucleic acid and the detection target nucleic acid is caused by one or more mutations selected from the group consisting of substitution, insertion, deletion, inversion, overlap and translocation or a combination thereof.
21. The kit according to claim 12 , wherein the target nucleic acid is a gene, and the difference in the base sequence of the gene to be the detection target relates to the onset and/or treatment sensitivity of a particular disease.