Reverse transcription with thermostable DNA polymerases - high temperature reverse transcription

• US 5,322,770 A
• Filed: 12/22/1989
• Issued: 06/21/1994
• Est. Priority Date: 12/22/1989
• Status: Expired due to Term

• Current Assignee: Roche Molecular Systems Inc (Roche Holding AG)
• Original Assignee: Hoffmann-La Roche Incorporated (Roche Holding AG)
• Inventors: Gelfand, David H.
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): ESCALLON, MIGUEL H
• Application Number:
  US07/455,611
• Time in Patent Office: 1,642 Days
• Field of Search: 435/6, 435/91, 435/172.3, 435/7.91, 536/18.7, 536/25.6, 935/77, 935/78
• US Class Current: 435/5
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12N 9/1276 RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)

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.

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.

2. A method according to claim 1 wherein said thermostable DNA polymerase having reverse transcriptase activity is Taq polymerase.

3. A method according to claim 2 wherein said temperature is between 50°

C. and 80°

C.

4. A method according to claim 3 wherein said buffer comprises a divalent cation from the group consisting of Mg²⁺ and Mn²⁺.

5. A method according to claim 4 wherein said divalent cation if Mn²⁺ at a concentration of between about 0.5 and 2.0 mM.

6. The method of claim 2 wherein said sample is a human biological sample.

7. The method of claim 6 wherein said primer is complementary to a specific RNA target molecule.

8. The method of claim 7 wherein said specific RNA target molecule encodes a cytokine.

9. The method of claim 8 wherein said cytokine is IL-δ

1.

10. The method of claim 7 wherein said specific RNA target molecule is diagnostic of a genetic or infectious disease.

11. A method for deleting a specific target RNA molecule in a sample suspected of containing said target RNA that comprises the step of:

(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.

12. The method for claim 11 wherein said thermostable DNA polymerase having reverse transcriptase activity is Taq polymerase.

13. The method of claim 12 that further comprises after step (b) and before step (c);

repeating steps (a) and (b) at least once.

14. The method of claim 12 wherein said specific RNA target molecule is diagnostic of a genetic or infectious disease.

15. A method for amplifying a target RNA molecular suspected of being present in a sample, the method comprising the steps of:

(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.

16. The method for RNA amplification of claim 15 wherein said thermostable DNA polymerase having reverse transcriptase activity is Taq polymerase.

17. The method of claim 16 wherein said RNA target is diagnostic of a genetic or infectious disease.

18. A method for preparing duplex cDNA from an RNA template that comprises the steps of:

(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.