Reverse transcription with thermostable DNA polymerase-high temperature reverse transcription

• US 5,310,652 A
• Filed: 06/24/1993
• Issued: 05/10/1994
• Est. Priority Date: 08/22/1986
• 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., Myers, Thomas W.
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): Escallon, Miguel H.
• Application Number:
  US08/082,182
• Time in Patent Office: 320 Days
• Field of Search: 435/6, 435/91, 435/227, 435/7.91, 436/21, 536/18.7, 536/25.6, 935/77, 935/78
• US Class Current: 435/6.11
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• 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: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• 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 1/703 Viruses associated with AIDS
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2527/101 Temperature
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2547/101 by confinement to a single tube/container

Abstract

Methods are provided for the replication and amplification of RNA sequences by thermoactive DNA polymerases. In a preferred embodiment, high temperature reverse transcription is coupled to nucleic acid amplification in a one tube, one enzyme procedure using a thermostable DNA polymerase. Methods for eliminating carry over contamination of amplifications due to prior reverse transcription reactions are also provided.

Claims

1. A homogeneous single addition method for copying a target RNA molecule in a sample to produce a cDNA, and then amplifying the resulting cDNA, the method comprising the steps of:

(a) treating said sample in a reaction mixture comprising a first and second primer, wherein said first primer is sufficiently complementary to said target RNA to hybridize therewith and initiate synthesis of a cDNA molecule complementary to said target RNA, and said second primer is sufficiently homologous to said target RNA to hybridize to said cDNA and initiate synthesis of an extension product, and a thermostable DNA polymerase having reverse transcriptase activity in the presence of all four deoxyribonucleoside triphosphates, in an appropriate buffer, wherein said buffer comprises Mn⁺², at a temperature sufficient for said thermostable DNA polymerase to initiate synthesis of an extension product of said first primer to provide a cDNA molecule complementary to said target RNA;

(b) treating said reaction mixture at an appropriate temperature to provide single-stranded cDNA;

(c) treating said reaction mixture at an appropriate temperature for said thermostable DNA polymerase to initiate synthesis of an extension product of said second primer to provide a double-stranded cDNA molecule; and

(d) amplifying the double-stranded cDNA molecule of step (c) by a polymerase chain reaction.

2. The method of claim 1 wherein said thermostable DNA polymerase is the Thermus aquaticus DNA polymerase or the Thermus thermophilus DNA polymerase.

3. The method of claim 2 wherein said DNA polymerase is recombinant Tth DNA polymerase.

4. The method of claim 1 wherein said sample comprises between 0.1 picograms and 1 microgram of RNA.

5. The method of claim 4 wherein said sample comprises between 1 and 10⁸ copies of said target RNA.

6. The method of claim 1 wherein the temperature at step (a) is between 40°

C. and 80°

C.

7. The method of claim 1 wherein said target RNA molecule is diagnostic of a genetic or infectious disease.