Nucleic acid probe molecule of hairpin-shape structure and method for detecting nucleic acids using the same

• US 6,114,121 A
• Filed: 11/13/1997
• Issued: 09/05/2000
• Est. Priority Date: 11/14/1996
• Status: Expired due to Fees

• Current Assignee: New Energy and Industrial Technology Development Organizatio
• Original Assignee: Aisin Cosmos R&D Co., Ltd. (Aisin Corp.)
• Inventors: Shigemori, Yasushi, Fujiwara, Jun
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): WHISENANT, ETHAN C
• Application Number:
  US08/969,320
• Time in Patent Office: 1,027 Days
• Field of Search: 435/6, 536/23.1, 536/24.3, 935/76, 935/77, 935/78
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2521/507 Recombinase
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2561/125 Ligase Detection Reaction [LDR]

Abstract

This invention provides a hairpin-shaped nucleic acid probe which is capable of forming stable hybrid with target nucleic acid, and a method for detecting nucleic acid containing a sequence complementary to a part of sequence of the probe. After contacting RecA to a hairpin-shaped single strand probe for hybridization, this complex is contacted with a target nucleic acid to form a triplex recombination intermediate using RecA function. After ligating the probe and the target nucleic acid, the target nucleic acid is detected by the label on the probe.

Claims

1. A method for detecting a target nucleic acid molecule having a certain nucleotide sequence, comprising:

preparing a nucleic acid probe molecule which comprises a first nucleotide portion with self-complementary stem-and-loop;

a second nucleotide portion with a single strand structure extending from one end of the first nucleotide portion; and

a detectable label bonded to the first or second nucleotide portion, said second nucleotide portion having a sequence complementary to that of at least an end portion of the target nucleic acid molecule to be detected, and said label providing an information that the target nucleic acid molecule is hybridized to the second nucleotide portion;

forming a probe-RecA complex by contacting RecA proteins to the nucleic acid probe molecule;

reacting the probe-RecA complex with a sample containing the double stranded target nucleic acid to form a hybrid in which said second nucleotide portion is hybridized with the end portion of said target nucleic acid molecule;

ligating said first nucleotide portion of the probe molecule to one end of said target nucleic acid molecule in the hybrid by using a ligase;

removing proteins including said RecA from said hybrid;

separating and removing any unhybridized probe molecule from said hybrid; and

detecting said label in said hybrid, thereby indicating the presence of said target nucleic acid molecule in said sample.

2. A method according to claim 1, wherein said ligase is an NAD requiring type ligase.

3. A method according to claim 2, wherein the ligase reaction is conducted in a solution at 37 to 65°

C.