Method for detecting guanine-abasic site in DNA

• US 10,370,703 B2
• Filed: 01/29/2015
• Issued: 08/06/2019
• Est. Priority Date: 03/14/2014
• Status: Active Grant

• Current Assignee: National Institute of Advanced Industrial Science and Technology (Government of Japan)
• Original Assignee: National Institute of Advanced Industrial Science and Technology (Government of Japan)
• Inventors: Wu, Chun
• Primary Examiner(s): Whisenant, Ethan C
• Application Number:
  US15/126,178
• Publication Number:
  US 20170088884A1
• Time in Patent Office: 1,650 Days
• CPC Class Codes: C12Q 1/44 involving esterase
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/119 incorporating abasic sites
• CPC Class Codes: C12Y 301/21 Endodeoxyribonucleases producing 5'-phosphomonoesters (3.1.21)
• CPC Class Codes: C12Y 301/21005 Type III site-specific deoxyribonuclease (3.1.21.5)

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.

Claims

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.