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

Claim 10:
11. 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-F667Y/CA, or (2) KlenTaq-s in combination with Taq/CA;

(iii) a plurality of primer pairs for separately amplifying said plurality of target sequences to produce a plurality of amplicons, wherein each primer pair comprises a respective 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 the blocked primer when the 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 the blocked primer, wherein said nucleic acid polymerase or a combination of nucleic acid polymerases is unable to remove said 2′,3′-dideoxyribonucleotide from the blocked primer when the 2′,3′-dideoxyribonucleotide does not anneal to said target sequence or said amplicon; and
(v) a plurality of fluorescent probes which are different from the blocked primers and complementary to said plurality of target sequences or corresponding amplicon, respectively, wherein each fluorescent probe comprises a first nucleotide attached to a fluorophore and a second nucleotide attached to a quencher, respectively, 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 the target sequence or amplicon during extension from the respective blocked primer, wherein said plurality of fluorescent probes comprises the same or different fluorophores;
subjecting said PCR reaction system to amplification reactions under appropriate reaction conditions; and
detecting the fluorescence signal of said PCR reaction system.