Method of purifying and analyzing nucleic acid

Patent metadata

Field	Value
Publication number	US-2025092453-A1
Application number	US-202418830268-A
Country	US
Kind code	A1
Filing date	Sep 10, 2024
Priority date	Sep 14, 2023
Publication date	Mar 20, 2025
Grant date	—

Abstract

The present invention relates to a method for purifying a nucleic acid and a method for analyzing the presence of a target nucleic acid in a sample, and relates to a method for purifying a target nucleic acid from a nucleic acid library to high purity or a method for analyzing the presence of a target nucleic acid in a sample. Specifically, the present invention relates to a method for purifying and analyzing a nucleic acid for selecting a target nucleic acid based on whether a nucleic acid complementary to the target nucleic acid is synthesized.

First claim

What is claimed is:

1 . A method of purifying a target nucleic acid, comprising:
(a) hybridizing a library containing the target nucleic acid with a primer that binds complementarily to the target nucleic acid; (b) reacting the hybridization reactant of step (a) with a preparation comprising (i) a nucleotide triphosphate (NTP) comprising an irreversible blocker and/or (ii) a blocker-free NTP selected from the group consisting of blocker-free ATP, CTP, GTP, and TTP that binds to the target nucleic acid; and (c) isolating the target nucleic acid to which the complementary nucleic acid is bound from the reactants of step (b).

2 . The method according to claim 1 , wherein, in the NTP comprising the irreversible blocker, the remaining NTPs except for one selected from the group consisting of the blocker-free ATP, CTP, GTP, and TTP may comprise an irreversible blocker.

3 . The method according to claim 2 , wherein step (b) comprises alternating NTPs comprising an irreversible blocker and blocker-free NTPs; or NTPs comprising an irreversible blocker and NTPs comprising a cleavable blocker for each reaction cycle.

4 . The method according to claim 1 , wherein the reaction in step (b) results in the binding of a nucleic acid complementary to the target nucleic acid and mismatches to the non-target nucleic acid, such that the reaction is stopped by an NTP comprising an irreversible blocker.

5 . The method according to claim 1 , wherein step (c) involves attaching a tag to the NTP containing an irreversible blocker and separating the product, in which the reaction has been halted by the NTP containing the irreversible blocker, from the target nucleic acid to which the complementary nucleic acid is bound.

6 . The method according to claim 1 , further comprising the step of (d) isolating only the target nucleic acid from the target nucleic acid to which the complementary nucleic acid is bound.

7 . The method according to claim 6 , wherein step (d) is performed by treating the target nucleic acid to which the complementary nucleic acid is bound with a nuclease.

8 . The method according to claim 1 , wherein the NTPs comprising the irreversible blocker of step (b) are dideoxy nucleoside triphosphates (ddNTPs) or pyrrolidinyl nucleoside triphosphates (prNTPs).

9 . The method according to claim 2 , wherein the blocker-free NTP of step (b) is a deoxy nucleoside triphosphate (dNTP).

10 . The method according to claim 2 , wherein the NTP comprising the cleavable blocker is one or more selected from the group consisting of 3′-O-azidomethyl-dNTP, 3′-ONH 2 -dNTP, 3′-O-allyl-dNTP, and 3′-O-2-nitrobenzyl-dNTP.

11 . The method according to claim 1 , wherein step (b) comprises an NTP comprising an irreversible blocker at a concentration of 20 to 200 μM.

12 . The method according to claim 1 , wherein the preparation of step (b) further comprises an enzyme, a buffer, and a salt.

13 . The method according to claim 11 , wherein the salt comprises MnCl 2 or MnSO 4 .

14 . The method according to claim 13 , wherein the salt comprises a concentration of 1 mM or more.

15 . A method for analyzing the presence of a target nucleic acid in a sample, comprising:
(a) hybridizing a sample containing the target nucleic acid with a primer that binds complementarily to the target nucleic acid; (b) reacting the hybridization reactant of step (a) with a preparation comprising (i) a nucleotide triphosphate (NTP) comprising an irreversible blocker and/or (ii) a blocker-free NTP selected from the group consisting of blocker-free ATP, CTP, GTP, and TTP that binds to the target nucleic acid; and (c) isolating the target nucleic acid to which the complementary nucleic acid is bound from the reactants of step (b).

16 . The method according to claim 15 , wherein, in the NTP comprising the irreversible blocker, the remaining NTPs except for one selected from the group consisting of the blocker-free ATP, CTP, GTP, and TTP may comprise an irreversible blocker.

17 . The method according to claim 16 , wherein step (b) comprises alternating NTPs comprising an irreversible blocker and blocker-free NTPs; or NTPs comprising an irreversible blocker and NTPs comprising a cleavable blocker for each reaction cycle.

18 . The method according to claim 15 , wherein the reaction in step (b) results in the binding of a nucleic acid complementary to the target nucleic acid and mismatches to the non-target nucleic acid, such that the reaction is stopped by an NTP comprising an irreversible blocker.

19 . The method according to claim 15 , wherein step (c) involves attaching a tag to the NTP containing an irreversible blocker and separating the product, in which the reaction has been halted by the NTP containing the irreversible blocker, from the target nucleic acid to which the complementary nucleic acid is bound.

20 . The method according to claim 15 , further comprising the step of (d) isolating only the target nucleic acid from the target nucleic acid to which the complementary nucleic acid is bound.

21 . The method according to claim 20 , wherein step (d) is performed by treating the target nucleic acid to which the complementary nucleic acid is bound with a nuclease.

22 . The method according to claim 15 , wherein the NTPs comprising the irreversible blocker of step (b) are dideoxy nucleoside triphosphates (ddNTPs) or pyrrolidinyl nucleoside triphosphates (prNTPs).

23 . The method according to claim 16 , wherein the blocker-free NTP of step (b) is a deoxy nucleoside triphosphate (dNTP).

24 . The method according to claim 16 , wherein the NTP comprising the cleavable blocker is one or more selected from the group consisting of 3′-O-azidomethyl-dNTP, 3′-ONH 2 -dNTP, 3′-O-allyl-dNTP, and 3′-O-2-nitrobenzyl-dNTP.

25 . The method according to claim 15 , wherein step (b) comprises an NTP comprising an irreversible blocker at a concentration of 20 to 200 μM.

26 . The method according to claim 15 , wherein the preparation of step (b) further comprises an enzyme, a buffer, and a salt.

27 . The method according to claim 26 , wherein the salt comprises MnCl 2 or MnSO 4 .

28 . The method according to claim 27 , wherein the salt comprises a concentration of 1 mM or more.

Assignees

• Gwangju Inst Science & Tech
• Atg Lifetech Inc

Inventors

• Choi Yeongjae
• Ryu Taehoon
• Park So Hyun
• Choi Eun Jin
• Kim Jihee

Classifications

• C12Q2527/137

  Concentration of a component of medium · CPC title

• C12Q2525/101

  incorporating non-naturally occurring nucleotides, e.g. inosine · CPC title

• C12Q2525/186

  incorporating a non-extendable or blocking moiety · CPC title

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q1/6813 Primary

  Hybridisation assays · CPC title