Reverse transcription with thermostable DNA polymerases - high temperature reverse transcription
First Claim
1. The method for reverse transcribing an RNA template that comprises treating a sample containing said RNA template with an oligonucleotide primer, which primer is sufficiently complementary to said RNA template to hybridize therewith, and a thermostable DNA polymerase having reverse transcriptase activity in the presence of all four deoxyribonucleotide triphosphate, in an appropriate buffer and at a temperature sufficient for said primer to hybridize to said RNA template and said thermostable DNA polymerase to catalyze the polymerization of said deoxyribonucleoside triphosphates to provide cDNA complementary to said RNA template.
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Accused Products
Abstract
Methods are provided for the replication and amplification of RNA sequences by thermostable DNA polymerases. Reverse transcription of RNA is catalyzed by, for example, 92 kDa Taq, 64 kDa Taq, and Tth DNA polymerase. Reverse transcription is coupled to PCR amplification in a one enzyme procedure using a thermostable polymerase.
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Citations
18 Claims
- 1. The method for reverse transcribing an RNA template that comprises treating a sample containing said RNA template with an oligonucleotide primer, which primer is sufficiently complementary to said RNA template to hybridize therewith, and a thermostable DNA polymerase having reverse transcriptase activity in the presence of all four deoxyribonucleotide triphosphate, in an appropriate buffer and at a temperature sufficient for said primer to hybridize to said RNA template and said thermostable DNA polymerase to catalyze the polymerization of said deoxyribonucleoside triphosphates to provide cDNA complementary to said RNA template.
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11. A method for deleting a specific target RNA molecule in a sample suspected of containing said target RNA that comprises the step of:
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(a) treating said sample with an oligonucleotide primer, which primer is sufficiently complementary to said target RNA to hybridize therewith, and a thermostable DNA polymerase having reverse transcriptase activity in the presence of all four deoxyribonucleotide triphosphate, in an appropriate buffer and at a temperature sufficient for said primer to hybridize to said target RNA and said thermostable DNA polymerase to catalyze the polymerization of said deoxyribonucleoside triphosphates to provide a cDNA sequence complementary to said target RNA; (b) treating said cDNA formed in step (a) to provide a single-stranded cDNA and a RNA template; and (c) determining whether said cDNA is present. - View Dependent Claims (12, 13, 14)
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15. A method for amplifying a target RNA molecular suspected of being present in a sample, the method comprising the steps of:
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(a) treating said sample with a first primer, which primer is sufficiently complementary to said target RNA to hybridize therewith, and a thermostable DNA polymerase having reverse transcriptase activity in the presence of all four deoxyribonycleoside triphosphates, in an appropriate buffer and at a temperature sufficient for said first primer to hybridize with said target RNA and said thermostable DNA polymerase to catalyze to polymerization of said deoxyribonucleoside triphosphates to provide cDNA complementary to said target RNA; (b) treating said cDNA formed in step (a) to provide single-stranded cDNA; (c) treating said single-stranded cDNA formed in step (b) with a second primer, wherein said second primer can hybridize to said single-stranded cDNA molecule and initiate synthesis of an extension product in the presence of a thermostable DNA polymerase under appropriate conditions to produce a double-stranded cDNA molecule; and (d) amplifying the double-stranded cDNA molecule of step (c) by a polymerase chain reaction. - View Dependent Claims (16, 17)
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18. A method for preparing duplex cDNA from an RNA template that comprises the steps of:
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(a) treating said RNA template with a first primer, which primer is sufficiently complementary to said RNA template to hybridize therewith, and a thermostable DNA polymerase having reverse transcriptase activity in the presence of all four deoxyribonucleoside triphosphates, in an appropriate buffer and at a temperature sufficient for said first primer to hybridize with said RNA template and said thermostable DNA polymerase to catalyze the polymerization of said deoxyribonucleoside triphosphates to provide cDNA complementary to said target RNA; (b) treating said cDNA formed in step (a) to provide single-stranded cDNA; (c) treating said single-stranded cDNA formed in step (b) with a second primer, wherein said second primer can hybridize to said single-stranded cDNA molecule and initiate synthesis of an extension product in the presence of a thermostable DNA polymerase under appropriate conditions to produce a double-stranded cDNA molecule.
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Specification