Patent ID: 11921049
Assignee: nan
Field: Measurement (Instruments)
Classification: CPC C  G | IPC C  G

Claim 0:
1. A plurality of pairs of forward and reverse 3′ blocked primers comprise deoxyribonucleotides for multiplex pyrophosphorolysis activated polymerization (PAP) to amplify a plurality of template nucleic acids in a reaction, comprising:
a) a first pair of forward and reverse 3′ blocked primers complementary to a first template nucleic acid, wherein the first forward or reverse primer comprises a first fluorophore covalently attached to a nucleotide in the internal region or at the 5′ end and a universal quencher is covalently attached to a first non-extendable, blocked nucleotide which lacks the 3′ hydroxyl group and is located at the 3′ end of the primer, and wherein once the primer anneals to its template nucleic acid, the first non-extendable, blocked nucleotide attached with the universal quencher is removed from the 3′ end of the primer by pyrophosphorolysis catalyzed by a polymerase to generate two products of 1) a 3′ unblocked primer which is covalently labeled with the first fluorophore in the internal region or at the 5′ end, and 2) a corresponding blocked nucleoside triphosphate which is covalently labeled with the universal quencher, thus the first fluorophore emits a fluorescence signal in PAP amplification, and
b) a second pair of forward and reverse 3′ blocked primers complementary to a second template nucleic acid, wherein the second forward or reverse primer comprises a second fluorophore covalently attached to a nucleotide in the internal region or at the 5′ end and a universal quencher is attached to a second non-extendable, blocked nucleotide which lacks the 3′ hydroxyl group and is located at the 3′ end of the primer, and wherein once the primer anneals to its template nucleic acid, the second non-extendable, blocked nucleotide covalently attached with the universal quencher is removed from the 3′ end of the primer by pyrophosphorolysis catalyzed by the polymerase to generate two products of 1) a 3′ unblocked primer which is covalently labeled with the second fluorophore in the internal region or at the 5′ end, and 2) a corresponding blocked nucleoside triphosphate which is covalently labeled with the universal quencher, thus the second fluorophore emits another fluorescence signal in PAP amplification.