Method for detecting guanine-abasic site in DNA

Patent metadata

Field	Value
Publication number	US-10370703-B2
Application number	US-201515126178-A
Country	US
Kind code	B2
Filing date	Jan 29, 2015
Priority date	Mar 14, 2014
Publication date	Aug 6, 2019
Grant date	Aug 6, 2019

Abstract

The present invention provides a method for detecting the presence or absence of a guanine-abasic site, the method being a process for detecting guanine opposite at least one abasic sites generated in a double-stranded DNA, comprising:
(1) step 1 of site-selectively cleaving at least one abasic sites in a double-stranded DNA using an enzyme; (2) step 2 of modifying the amino group at position 2 of guanine opposite the abasic sites using a modifier; and (3) step 3 of performing polymerase chain reaction on the modified double-stranded DNA obtained by conducting step 1 and step 2, which serves as a template, to search for the presence or absence of an amplification product, the sequence of steps 1 and 2 being not limited to the order presented.

First claim

The invention claimed is:

1. A method for detecting the position of a guanine-abasic site generated in a double-stranded DNA, comprising:
(1) biotinylating an unpaired guanine residue in a double-stranded DNA containing at least one abasic site and the unpaired guanine;
(2) purifying the biotinylated double-stranded DNA on an avidin column;
(3) performing polymerase chain reaction on the purified biotinylated double-stranded DNA, which serves as a template and obtaining an amplification product;
(4) searching for a mutation from guanine to adenine in a base sequence of the amplification product; and
(5) detecting the position of the mutation from guanine to adenine as the position of the guanine-abasic site.

2. The method according to claim 1 , wherein biotinylating the unpaired guanine residue in step (1) is performed by
(i) introducing an acetylene group into the guanine residue of the guanine-abasic site, and
(ii) reacting the resultant with a reagent containing a biotin group and an azido group.

3. The method according to claim 2 , wherein the reagent for introducing an acetylene group is 2-Oxohex-5-ynal.

4. The method according to claim 2 , wherein the reagent containing a biotin group and an azido group is Azido-PEO3-Biotin.

5. The method according to claim 1 , wherein non-biotinylated double-stranded DNA is removed in step (2).

6. The method according to claim 1 , wherein polymerase chain reaction in step (3) is performed using Taq DNA polymerase.

7. The method according to claim 1 wherein searching for a mutation from guanine to adenine in step (4) is performed by DNA sequencing.

Assignees

• Aist

Inventors

• Wu Chun

Classifications

• C12Y301/21005

  Type III site-specific deoxyribonuclease (3.1.21.5) · CPC title

• C12Q2525/119

  incorporating abasic sites · CPC title

• C12Q1/44 Primary

  involving esterase · CPC title

• C12Y301/21

  Endodeoxyribonucleases producing 5'-phosphomonoesters (3.1.21) · CPC title

• C12Q1/6869

  Methods for sequencing · CPC title