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US 12,247,230 B2Granted
Mutant polymerases and uses thereof
AssigneeDna Polymerase Technology, Inc.
InventorsWayne M. Barnes, Milko B. Kermekchiev, Zhian Zhang
Life of this patent
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Abstract
Provided are mutant polymerases having DNA polymerase activity and reverse transcriptase activity or strand displacement activity, along with target nucleic acid amplification methods employing such mutant polymerases.
Claims15
A method of synthesizing a complementary DNA (cDNA) in a reverse transcriptase reaction comprising:
- forming an assay mixture comprising
- (A) a sample comprising a target RNA,
- (B) a primer which anneals to the target RNA,
- (C) a buffer, and
- (D) at least one mutant polymerase comprising a polypeptide sequence having
- (i) at least 95% sequence identity to SEQ ID NO:
- 1 or SEQ ID NO:
- 2,
- (ii) reverse transcriptase activity and DNA polymerase activity, and
- (iii) 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
- allowing reverse transcription of the target RNA to synthesize the cDNA in the assay mixture, 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.
10 more elements
A method of amplifying a target nucleic acid in a reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) comprising:
- forming an assay mixture comprising
- (A) a sample comprising the target nucleic acid, wherein the target nucleic acid is a target RNA,
- (B) four or six LAMP primers, wherein at least one primer anneals to the target RNA and wherein the four to six LAMP primers anneal to the cDNA or DNA synthesized therefrom,
- (C) a buffer, and
- (D) at least one mutant polymerase comprising a polypeptide sequence having
- (a) at least 95% sequence identity to SEQ ID NO:
- 1 or SEQ ID NO:2,
- (b) DNA polymerase activity, reverse transcriptase activity, and strand displacement activity, and
- (c) 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
- amplifying the target nucleic acid in the assay mixture in RT-LAMP, wherein the assay mixture for 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.
9 more elements
Dependencies are inferred from the claim text.