Patent ID: 11879153
Assignee: BIOVUE TECHNOLOGY LTD
Field: Biotechnology (Chemistry)
Classification: CPC C | IPC C

Claim 0:
1. A method for detecting nucleic acids by fluorescent PCR, comprising:
preparing a PCR reaction system, said PCR reaction system comprising:
(i) a nucleic acid sample comprising or suspected of comprising a target sequence;
(ii) a nucleic acid polymerase or a combination of nucleic acid polymerases, having a 5′→3′ polymerase activity, a 5′→3′ exonuclease activity, and a pyrophosphatase activity,
wherein the nucleic acid polymerase or the combination of nucleic acid polymerases is (1) Taq DNA polymerase with the F667Y mutation which is modified with citraconic anhydride (Taq-F667Y/CA), or (2) Taq DNA polymerase with N fragment truncated and the F667Y mutation (KlenTaq-s) in combination with a Taq DNA polymerase modified with citraconic anhydride (Taq/CA);

(iii) a primer pair for amplifying said target sequence to produce an amplicon, said primer pair comprising at least one blocked primer which comprises a 2′,3′-dideoxyribonucleotide located at the 3′ end of the blocked primer, wherein said 2′,3′-dideoxyribonucleotide blocks the extension of said nucleic acid polymerase or combination of nucleic acid polymerases when said 2′,3′-dideoxyribonucleotide does not anneal to said target sequence or said amplicon,
(iv) pyrophosphate, wherein in the presence of the pyrophosphate said nucleic acid polymerase or a combination of nucleic acid polymerases is capable of using the pyrophosphatase activity to remove said 2′,3′-dideoxyribonucleotide from said blocked primer when said 2′,3′-dideoxyribonucleotide anneals to said target sequence or said amplicon, allowing said nucleic acid polymerase or combination of nucleic acid polymerases to use the 5′→3′ polymerase activity to extend from said blocked primer, wherein said nucleic acid polymerase or a combination of nucleic acid polymerases is unable to remove said 2′,3′-dideoxyribonucleotide from said blocked primer when said 2′,3′-dideoxyribonucleotide does not anneal to said target sequence or said amplicon; and
(v) a fluorescent probe which is different from the blocked primer and complementary to the target sequence or the amplicon, wherein said fluorescent probe comprises a first nucleotide linked to a fluorophore and a second nucleotide linked to a quencher, wherein the fluorescence signal of said fluorophore is quenched by said quencher when said first nucleotide is not hydrolyzed from said fluorescent probe, and wherein said nucleic acid polymerase or combination of nucleic acid polymerases is capable of using the 5′→3′ exonuclease activity to hydrolyze said first nucleotide from said fluorescent probe bound to said target sequence or said amplicon during extension from the blocked primer such that the fluorescence signal of said fluorophore is not quenched by said quencher;
subjecting said PCR reaction system to amplification reactions under appropriate reaction conditions; and
detecting the fluorescence signal of said PCR reaction system.