Method for detecting nucleic acid mutation by detecting chemiluminiscence generated with by-product of complementary strand extension reaction

• US 6,750,018 B2
• Filed: 09/05/2001
• Issued: 06/15/2004
• Est. Priority Date: 09/28/2000
• Status: Expired due to Term

• Current Assignee: Hitachi, Ltd.
• Original Assignee: Hitachi, Ltd.
• Inventors: Kamahori, Masao, Kambara, Hideki, Okano, Kazunori, Zhou, Guohua
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US09/945,703
• Publication Number:
  US 20030049628A1
• Time in Patent Office: 1,014 Days
• Field of Search: 435/6, 435/91.1, 435/91.2
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer

Abstract

To provide a method and an apparatus for detecting DNA mutation without using electrophoresis, the presence or absence of specific sequence is detected by a method comprising a step of serially hybridizing a 5-base-long to 8-base-long short oligomer 6, which is capable of being engaged in the complementary strand extension reaction, and a long oligomer 61, which is capable of hybridizing with a target DNA but incapable of being engaged in the complementary strand extension reaction, with the target DNA 63; a step of carrying out the complementary strand extension reaction 66 starting from the short primer using four kinds of nucleic acid substrates and polymerase; and a step of detecting photo-emission, in which pyrophosphate 68 produced as a reaction by-product of the complementary strand extension reaction is converted into ATP and the photo-emission reaction is carried out using an enzyme.

Claims

1. A method for detecting nucleic acid mutation comprising:

hybridizing a primer, containing a complementary sequence complementary to a region of a specific sequence of target nucleic acid and a base not complementary to the target nucleic acid at second base or third base from 3′

end of the primer, with the target nucleic acid;

performing complementary strand extension reaction from the primer using polymerase, plurality of nucleic acid substrates and the primer;

generating chemoluminiscence using ATP and an enzyme, the ATP converted from pyrophosphate generated as by-product of the complementary strand extension reaction; and

detecting the chemoluniniscence generated in the step of generating chemoluninscence wherein the specific sequence of target nucleic acid is related to mutation of the nucleic acid and presence or absence of the specific sequence of target nucleic acid is detected;

wherein extended complementary strand generating in the step of performing complementary strand extension reaction is decomposed with an enzyme, the primer being repeatedly hybridized with the target nucleic acid to perform the complementary strand extension reaction.

2. A method for detecting nucleic acid mutation comprising:

hybridizing a primer, containing a complementary sequence complementary to a region of a specific sequence of target nucleic acid and a base not complementary to the target nucleic acid at second base or third base from 3′ and

of the primer, with the target nucleic acid;

performing complementary strand extension reaction from the primer using polymerase, plurality of nucleic acid substrates and the primer;

generating chemoluniscence using ATP and an enzyme, the ATP converted from pyrophosphate generated as by-product of the complementary strand extension reaction; and

detecting the chemoluminiscence generated in the step of generating chemoluniscence wherein the specific sequence of target nucleic acid is related to mutation of the nucleic acid and presence or absence of the specific sequence of target nucleic acid is detected;

wherein extended complementary strand generating in the step of performing complementary strand extension reaction is decomposed with an enzyme, the primer being repeatedly hybridized with the target nucleic acid to perform the complementary strand extension reaction; and

wherein the enzyme decompose the extended complementary strand from 5′

end of the extended complementary strand.