Patent ID: 9133491
Filing Date: 2015-09-15
Classification: C12N,C12P,C12Q

Abstract:
1. A method for synthesizing nucleic acid on a RNA template, comprising: (a) annealing to the RNA template a complementary 3′ blocked primer wherein the 3′ blocked primer has a dideoxynucleotide at the 3′ end, which, if not removed, would prevent the 3′ blocked primer from being extended; (b) removing the dideoxynucleotide from the 3′ end of the 3′ blocked primer by pyrophosphorolysis to produce a 3′ unblocked primer; and (c) extending the 3′ unblocked primer by polymerization to produce a nucleic acid product, wherein steps (b) and (c) are catalyzed by a mutant Taq DNA polymerase having RNA-dependent DNA polymerase activity, and wherein the mutant DNA polymerase has a 667Y mutation and another mutation selected from the group consisting of 599V, 602A, 605A, 608V, 681G, 681K, 681V, 681R, 742S and 747I mutations corresponding to Taq DNA polymerase according to SEQ ID NO: 10.