CPC C12N 9/1252 (2013.01) [C12N 9/1276 (2013.01); C12N 15/1096 (2013.01); C12Q 1/6846 (2013.01); C12Y 207/07007 (2013.01); C12Y 207/07049 (2013.01); C12Q 2521/101 (2013.01); C12Q 2531/101 (2013.01); C12Q 2531/119 (2013.01)] | 17 Claims |
1. A method of amplifying a target nucleic acid in a reverse transcriptase polymerase chain reaction (RT-PCR) comprising:
(i) forming an assay mixture comprising
(A) a sample comprising the target nucleic acid, wherein the target nucleic acid is a target RNA,
(B) a primer which anneals to the target RNA and its DNA equivalent and a primer which anneals to cDNA transcribed from the target RNA,
(C) a buffer, and
(D) at least one mutant polymerase comprising a polypeptide sequence having
at least 95% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2,
reverse transcriptase activity and DNA polymerase activity, and
a D732N substitution according to the numbering of wild type Taq polymerase of SEQ ID NO: 1, wherein the assay mixture does not include a separate reverse transcriptase enzyme in an amount sufficient to generate cDNA from the target RNA or in an amount sufficient to initiate reverse transcription of the target RNA; and
(ii) amplifying the target nucleic acid in the assay mixture in RT-PCR, wherein the amplifying does not include a separate reverse transcriptase enzyme in an amount sufficient to generate cDNA from the target RNA or in an amount sufficient to initiate reverse transcription of the target RNA.
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