Improved multiplex nucleic acid amplification using blocked primers
AssigneeBiomerieux
InventorsPaul van de Wiel, Birgit Deiman, Dianne van Strijp
Life of this patent
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Abstract
The present invention is in the field of nucleic acid amplification, and in particular in transcription-based amplification, providing improvements thereof. Specifically, the present invention provides primers, and methods for using them, that improve transcription-based amplification reactions, in particular multiplex reactions.
Claims25
A method of performing a specific transcription-based amplification reaction of a selected first target nucleic acid in a sample, in a reaction wherein reagents specific for amplification of more than one target nucleic acid are present, comprising:
- (1) contacting the sample with
- (a) a first promoter oligonucleotide having a 5′ end and a 3′ end, the first promoter oligonucleotide comprising, at the 5′end, an RNA polymerase promoter sequence, and 3′ of the RNA polymerase promoter sequence, a sequence complementary to a first target portion of the first target nucleic acid, and
- (b) a second promoter oligonucleotide having a 5′ end and a 3′ end, the second promoter oligonucleotide comprising, at the 5′end, an RNA polymerase promoter sequence, and 3′ of the RNA polymerase promoter sequence, a sequence complementary to a first target portion of a second target nucleic acid wherein further, the second promoter oligonucleotide comprises, at the 3′ end, a blocking moiety such that extension therefrom is essentially prohibited;
- and
- (2) providing selected reagents and conditions for transcription-based amplification.
A method of performing a specific transcription-based amplification reaction of a selected first target nucleic acid in a sample, in a reaction wherein more than one target nucleic acid, if present in the sample, is to be amplified, comprising:
- (1) contacting the sample with
- (a) a first promoter oligonucleotide having a 5′ end and a 3′ end, the first promoter oligonucleotide comprising, at the 5′end, an RNA polymerase promoter sequence, and 3′ of the RNA polymerase promoter sequence, a sequence complementary to a first target portion of the first target nucleic acid, and
- (b) a second promoter oligonucleotide having a 5′ end and a 3′ end, the second promoter oligonucleotide comprising, at the 5′end, an RNA polymerase promoter sequence, and 3′ of the RNA polymerase promoter sequence, a sequence complementary to a first target portion of a second target nucleic acid wherein further, the second oligonucleotide comprises, at the 3′ end, a blocking moiety such that extension therefrom is prohibited;
- and
- (2) providing selected reagents and conditions for transcription-based amplification.
A composition for performing multiplex hybridization to and amplification of two or more target nucleic acids comprising two or more promoter oligonucleotides, each having a 5′ end and a 3′ end, a first promoter oligonucleotide comprising, at the 5′end, an RNA polymerase promoter sequence, and 3′ of the RNA polymerase promoter sequence, a sequence complementary to a first target portion of the first target nucleic acid, and a second promoter oligonucleotide having a 5′ end and a 3′ end, the second promoter oligonucleotide comprising, at the 5′end, an RNA polymerase promoter sequence, and 3′ of the RNA polymerase promoter sequence, a sequence complementary to a first target portion of a second target nucleic acid wherein further, the second promoter oligonucleotide comprises, at the 3′ end, a blocking moiety such that extension therefrom is essentially prohibited.
Dependencies are inferred from the claim text.