US 9,845,495 B2Granted

Method and kit for detecting target nucleic acid

AssigneesAbbott Laboratories, Tokyo Institute of Technology

InventorKen Komiya

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Abstract

[Problem] To provide a method for detecting a nucleic acid (such as DNA and RNA) under isothermal conditions, in particular a method by which a short-chain nucleic acid can be directly detected. [Solution] A method for detecting a target nucleic acid in a sample of the present invention comprises: (a) a step of preparing a first oligonucleotide which comprises, in the direction from 5′ to 3′, a first arbitrary sequence, an endonuclease recognition site that is used in a nicking reaction, and a sequence complementary to the target nucleic acid; (b) a step of carrying out a nucleic acid amplification reaction using the target nucleic acid contained in the sample as a primer in the presence of an endonuclease which recognizes the endonuclease recognition site that is used in a nicking reaction; and (c) a step of detecting an oligonucleotide which is obtained by the nucleic acid amplification reaction and comprises a sequence complementary to the first arbitrary sequence.

Claims18

Dependent claims

A method for detecting a target nucleic acid in a sample, the method comprising:

(a) preparing a first oligonucleotide comprising, in a direction from 5′ to 3′, (i) a first arbitrary sequence having about 5 to about 50 nucleotides, (ii) an endonuclease recognition site that is used in a nicking reaction, and (iii) a sequence complementary to the 3′ end of the target nucleic acid;
and a second oligonucleotide comprising, in a direction from 5′ to 3′, (i) a second arbitrary sequence having about 5 to about 50 nucleotides and a sequence substantially homologous to the first arbitrary sequence, (ii) an endonuclease recognition site that is used in a nicking reaction, and (iii) a sequence substantially homologous to the first arbitrary sequence;
(b) preparing a reaction mixture comprising the sample, the first oligonucleotide, the second oligonucleotide, a polymerase, and an endonuclease;
(c) maintaining the reaction mixture at a temperature of about 35° C. to about 37° C. under conditions wherein the 3′ end of a target nucleic acid binds to the sequence of the first oligonucleotide complementary to the 3′ end of the target nucleic acid and primes replication to produce an extended target nucleic acid which is nicked by the endonuclease to produce a nick which further primes replication to produce a signal DNA complementary to the second arbitrary sequence of the second oligonucleotide;
and
wherein said signal DNA binds to the second oligonucleotide at the 3′ end sequence substantially homologous to the first arbitrary sequence and primes replication to produce an extended signal DNA which is nicked by the endonuclease to produce a nick which further primes replication to produce additional signal DNA;
(d) detecting the signal DNA, wherein the presence of the signal DNA detects the target nucleic acid.

4 more elements

A method for detecting a target nucleic acid in a sample, said method comprising combining said sample with:

(a) a first oligonucleotide comprising, in a 5′ to 3′ direction, (i) a first arbitrary sequence having about 5 to about 50 nucleotides, (ii) an endonuclease recognition site, and (iii) a sequence complementary to the 3′ end of said target nucleic acid;
(b) a second oligonucleotide comprising, in a 5′ to 3′ direction, (i) a second arbitrary sequence having about 5 to about 50 nucleotides substantially homologous to said first arbitrary sequence, (ii) an endonuclease recognition site, and (iii) a sequence substantially homologous to the first arbitrary sequence;
(c) a polymerase;
and
(d) at least one endonuclease for a nicking reaction;
maintaining a temperature of about 35° C. to about 37° C. under conditions wherein the 3′ end of the target nucleic acid binds to the sequence of the first oligonucleotide complementary to the 3′ end of the target nucleic acid and primes replication to produce an extended target nucleic acid which is nicked by the endonuclease to produce a nick which further primes replication to produce a signal DNA complementary to the second arbitrary sequence of the second oligonucleotide;
and
wherein said signal DNA binds to the second oligonucleotide at the 3′ end sequence substantially homologous to the first arbitrary sequence and primes replication to produce an extended signal DNA which is nicked by the endonuclease to produce a nick which further primes replication to produce additional signal DNA;
and
detecting the signal DNA, wherein the presence of the signal DNA detects the target nucleic acid.

7 more elements

Dependencies are inferred from the claim text.

Facts

Application: 13/992,058Filed 12 Dec 2011
Priority: JP 2010-27594810 Dec 2010
Published: US 2014/0017692 A116 Jan 2014
Granted: 19 Dec 2017
Term ends: Not estimatedThe source records don’t support an estimate.

Classification4 codes

C12Q 1/6844
Involving nucleic acids: nucleic acid amplification reactions
C12Q 1/6816
Hybridisation assays: characterised by the detection means
C12Q 1/682
Characterised by the detection means: signal amplification
C12Q
Measuring or testing processes involving enzymes, nucleic acids or microorganisms