Thermostable purified reverse transcriptase and methods of use
View Patent ↗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.
1 . 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 . The method for reverse transcription of claim 1 , wherein the reverse transcriptase comprises a DNA polymerase A domain that has an amino acid sequence with more than 95% sequence identity to DNA polymerase A domain of SEQ ID NO: 8, and wherein the reverse transcriptase activity is heat-inactivated.
3 . The method for reverse transcription of claim 1 , wherein step of heat inactivating the reverse transcriptase is carried out at a temperature from 83° C. to 95° C.
4 . The method for reverse transcription of claim 1 , wherein the step of reverse transcription is performed at a temperature from 55° C. to 70° C.
5 . The method for reverse transcription of claim 1 , wherein the reverse transcriptase has an optimal temperature from 60° C. to 62° C. for catalyzing reverse transcription.
6 . The method for reverse transcription of claim 1 , wherein the method further comprises (d) using a second DNA-dependent DNA polymerase to catalyze DNA-dependent DNA polymerization using the reverse-transcribed DNA as template.
7 . The method for reverse transcription of claim 1 , wherein the reverse transcriptase contains a tyrosine to phenylalanine substitution corresponding to position 420 of SEQ ID NO: 8.
8 . 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.
9 . The method for RNA-dependent DNA pyrophosphorolysis of claim 8 , wherein the reverse transcriptase comprises a DNA polymerase A domain that has an amino acid sequence with more than 90% sequence identity to DNA polymerase A domain of SEQ ID NO: 8.
10 . The method for RNA-dependent DNA pyrophosphorolysis of claim 8 , wherein the reverse transcriptase contains a phenylalanine to tyrosine substitution at amino acid position 420 of SEQ ID NO: 8.
11 . The method for RNA-dependent DNA pyrophosphorolysis of claim 8 , wherein the method further comprises steps (d): catalyzing reverse transcription on the RNA template with the reverse transcriptase to generate a reverse-transcribed DNA, and then (e) heat-inactivating the reverse transcriptase activity.
12 . The method for RNA-dependent DNA pyrophosphorolysis of claim 8 , wherein the step of heat inactivating the reverse transcriptase activity is carried out at a temperature >83° C.
13 . The method for RNA-dependent DNA pyrophosphorolysis of claim 8 , wherein the step of heat inactivating the reverse transcriptase is carried out at a temperature from 83° C. to 95° C.
14 . The method for RNA-dependent DNA pyrophosphorolysis of claim 8 , wherein the step of RNA-dependent DNA pyrophosphorolysis is carried out at a temperature from 55° C. to 70° C.
15 . The method for RNA-dependent DNA pyrophosphorolysis of claim 8 , wherein the reverse transcriptase has an optimal temperature from 60° C. to 62° C. for catalyzing RNA-dependent DNA pyrophosphorolysis.
16 . The method for RNA-dependent DNA pyrophosphorolysis of claim 11 , wherein the method further comprises:
(f) a second 3′ blocked primer that has a non-extendable nucleotide at the 3′ blocker anneals to the reverse-transcribed DNA template;
(g) a second DNA-dependent DNA polymerase that catalyzes DNA-dependent DNA pyrophosphorolysis to remove the 3′ blocker from the 3′ blocked primer to activate the primer; and
(h) the second DNA-dependent DNA polymerase that catalyzes DNA-dependent DNA polymerization using the reverse-transcribed DNA as template.
17 . The method for RNA-dependent DNA pyrophosphorolysis of claim 16 wherein the steps (a) to (h) are conducted in a single reaction tube.