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US 12,692,525 B1Granted
Thermostable purified reverse transcriptase and methods of use
OwnerShaofeng Ding, Qiang Liuinventors
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Abstract
Thermophilic purified Type A DNA-dependent DNA polymerases efficiently catalyze reverse transcription and RNA-dependent DNA pyrophosphorolysis using ribonucleic acid (RNA) templates, virtually also being reverse transcriptases. At optimal temperatures of 60° C. to 62° C., the thermophilic reverse transcriptases are particularly efficient for reverse transcription in RT-PCR and RT-PAP amplifications.
Claims17
A method for reverse transcription using RNA-dependent DNA polymerization comprising:
- (a) providing a thermophilic purified DNA-dependent DNA polymerase of Carboxydothermus pertinax that catalyzes reverse transcription by RNA-dependent DNA polymerization on an RNA template, wherein the polymerase is also a reverse transcriptase and an RNA-dependent DNA polymerase comprising an amino acid sequence that has more than 95% sequence identity to DNA-dependent DNA polymerase of SEQ ID NO:
- 8;
- (b) catalyzing reverse transcription on an RNA template with the reverse transcriptase to generate a reverse-transcribed DNA;
- and then, (c) heat-inactivating the reverse transcriptase activity ≥83° C.;
- wherein the reverse transcriptase is >90% inactivated after 2 minutes at 83° C.
2 more elements
A method for RNA-dependent DNA pyrophosphorolysis comprising:
- (a) providing a thermophilic purified DNA-dependent DNA polymerase that catalyzes RNA-dependent DNA pyrophosphorolysis on an RNA template, wherein the polymerase is also a reverse transcriptase and an RNA-dependent DNA polymerase comprising an amino acid sequence that has more than 85% sequence identity to DNA-dependent DNA polymerase of SEQ ID NO:
- 8;
- (b) providing a 3′ blocked primer that has a non-extendable nucleotide at the 3′-blocker to anneal to an RNA template;
- and
- (c) catalyzing RNA-dependent DNA pyrophosphorolysis with the reverse transcriptase to remove the 3′ blocker from the 3′ blocked primer.
2 more elements
Dependencies are inferred from the claim text.