Nucleic acid amplification employing ligatable hairpin probe and transcription
First Claim
1. 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 hairpin-forming self-complementary sequence, said target sequence is transcribable in the presence of an RNA polymerase and required ribonucleoside triphosphates under said hairpin-forming hybridizing conditions.
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Abstract
Specific nucleic acid sequences are amplified through the use of a hairpin probe which, upon hybridization with and ligation to, a target sequence is capable of being transcribed. The probe comprises a single stranded self-complementary sequence which, under hybridizing conditions, forms a hairpin structure having a functional promoter region, and further comprises a single stranded probe sequence extending from the 3'"'"' end of the hairpin sequence. Upon hybridization with a target sequence complementary to the probe sequence and ligation of the 3'"'"' end of the hybridized target sequence to the 5'"'"' end of the hairpin probe, the target sequence is rendered transcribable in the presence of a suitable RNA polymerase and appropriate ribonucleoside triphosphate (rNTPs). Amplification is accomplished by hybridizing the desired target nucleic acid sequence with the probe, ligating the target sequence to the probe, adding the RNA polymerase and rNTPs to the separated hybrids, and allowing transcription to proceed until a desired amount of RNA transcription product has accumulated. The amplification method is particularly useful in assays for the detection of particular nucleic acid sequences.
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40 Claims
- 1. 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 hairpin-forming self-complementary sequence, said target sequence is transcribable in the presence of an RNA polymerase and required ribonucleoside triphosphates under said hairpin-forming hybridizing conditions.
Specification