Nucleic acid amplification employing ligatable hairpin probe and transcription

Claim 2

A nucleic acid probe comprising (1) a single stranded self-complementary sequence capable of forming, under hybridizing conditions, a hairpin structure having a functional promoter region, and (2) a single stranded probe sequence extending from the 3′ end of said self-complementary sequence, whereby upon hybridization of a target sequence complementary to said probe sequence and ligation of the resulting hybridized target sequence to the 5′ end of the haiprin-forming self-complementary sequence said target sequence is transcribable in the presence of an RNA polymerase and required rihonucleoside triphosphates under said hairpin-forming hyhridizing conditions.

Claim 3

The probe of claim 1 composed of DNA and wherein said polymerase is a DNA-dependent RNA polymerasel,preferably T7, T3, or SP6 bacteriophage RNA polymerase, and preferably wherein said promoter region comprises the following bare sequence shown in its hairpin form: wherein n is an integer from 2 to 50 inclusive.

Claim 4

A method for amplifying a particular target nucleic acid sequence, comprising the steps of: (a) hybridizing said target sequence with a probe of claim 1 in a liquid mixture, (b) ligating the resulting hybridized target sequence to the 5′ end of said hairpin-forming self-complementary sequence, (c) adding to the ligated hybrids said RNA polymerase and ribonucleoside triphosphates sufficient for transcription of said target sequence in the hybrids, and (d) allowing the resulting transcription of said target sequence to take place for predetermined period of time to accumulate the resulting complementary RNA transcription product as an amplification of said target sequence.

Claim 5

method of claim 3 wherein the resulting RNA transcription product is further amplified in a second stage amplification.

Claim 7

A method for detecting a particular target nucleic acid sequence in a test sample, comprising the steps of: (a) hybridizing said target sequence in the test sample with a probe of claim 1 in a liguid mixture, (b) ligating the resulting hybridized target sequence to the 5′ end of said hairpin-forming self-complementary sequence, (c) adding to the ligated sequences said RNA polymerase and ribonucleoside triphosphates sufficient for transcription of said target sequence in the hybrids, (d) allowing the resulting transcription of said target sequence to take place for a predetermined period of time to accumulate the resulting complementary RNA transcription product, and (e) detecting said accumulated transcripcion product.

Claim 8

The method of claim 6 wherein the resulting RNA transcription product is further amplified in a second stage amplification.

Claim 10

A rengent kit for use in amplifying a particular nucleic acid sequence, comprising: (1) a nucleic acid probe of any of claims 1 and 2 which upon hybridization with and ligation to said target sequence, is transcribable in the presence of an RNA polymerase and ribonucleoside triphosphates, and (2) said RNA polymerase.