US 11,814,655 B2
Mutant polymerases and uses thereof
Wayne M. Barnes, University City, MO (US); Milko B. Kermekchiev, St. Louis, MO (US); and Zhian Zhang, Ballwin, MO (US)
Assigned to DNA POLYMERASE TECHNOLOGY, INC., St. Louis, MO (US)
Filed by DNA POLYMERASE TECHNOLOGY, INC., St. Louis, MO (US)
Filed on May 28, 2021, as Appl. No. 17/303,445.
Application 17/303,445 is a continuation of application No. 15/572,693, granted, now 11,091,745, previously published as PCT/US2016/032041, filed on May 12, 2016.
Claims priority of provisional application 62/160,404, filed on May 12, 2015.
Prior Publication US 2021/0348142 A1, Nov. 11, 2021
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 9/12 (2006.01); C12Q 1/6844 (2018.01); C12N 15/10 (2006.01)
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.