High temperature reverse transcription using mutant DNA polymerases

• US 7,179,590 B2
• Filed: 03/30/2001
• Issued: 02/20/2007
• Est. Priority Date: 04/18/2000
• Status: Expired due to Term

• Current Assignee: Roche Molecular Systems (Roche Holding AG)
• Original Assignee: Roche Molecular Systems (Roche Holding AG)
• Inventors: Gelfand, David Harrow, Higuchi, Russell Gene, Myers, Thomas William, Smith, Edward Soh, Elfstrom, Carita Maria, Wang, Alice Ming, Schonbrunner, Nancy Jeneane
• Primary Examiner(s): GOLDBERG, JEANINE ANNE
• Application Number:
  US09/823,649
• Publication Number:
  US 20020012970A1
• Time in Patent Office: 2,153 Days
• Field of Search: 435/6, 435/91.1, 530/300, 530/327, 536/23.1, 536/23.2
• US Class Current: 435/6.1
• 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 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2527/101 Temperature
• CPC Class Codes: C12Q 2547/101 by confinement to a single tube/container

Abstract

The present invention relates to improved reverse transcription methods using a modified thermostable DNA polymerases, particularly in a magnesium ion buffer. These methods are particularly useful in combined reverse-transcription/amplification reactions.

Claims

1. A method for reverse transcribing an RNA, that comprises:

(a) providing a reverse transcription reaction mixture comprising said RNA, a primer, Mg+2, and a mutant thermoactive DNA polymerase, wherein said mutant DNA polymerase is characterized in that

i) in its native form said DNA polymerase comprises a polymerase domain comprising an amino acid sequence that is SEQ ID NO;

1, wherein said polymerase domain has the ability to incorporate nucleotides;

ii) the amino acid at position 2 of said amino acid sequence is S or A and the amino acid at position 5 of said amino acid sequence is L or I; and

iii) the amino acid at position 4 of said amino acid sequence is mutated in comparison to said native sequence to an amino acid other than E, A, G, or P; and

(b) treating said reaction mixture at a temperature sufficient for said mutant DNA polymerase to initiate synthesis of an extension product of said primer to provide a cDNA molecule complementary to said RNA.

2. The method of claim 1, wherein said mutant DNA polymerase in its native form comprises an amino acid sequence that is SEQ ID NO:

2, the amino acid at position 3 of said amino acid sequence is Q or G, and the amino acid at position 6 of said amino acid sequence is S or A.

3. The method of claim 1, wherein said mutant DNA polymerase in its native form comprises a polymerase domain comprising an amino acid sequence that is SEQ ID NO:

3.

4. The method of claim 1, wherein said mutant DNA polymerase in its native form comprises a polymerase domain comprising an amino acid sequence that is SEQ ID NO:

4, and the amino acid at position 3 is Q or G.

5. The method of claim 1, wherein said mutant DNA polymerase is thermostable.

6. The method of claim 1, wherein said mutant DNA polymerase is a mutant form of a Thermus species DNA polymerase.

7. The method of claim 1, wherein said mutant DNA polymerase is a mutant form of Thermus thermophilus DNA polymerase or Thermus aquaticus DNA polymerase.

8. The method of claim 1, wherein said temperature of said reaction mixture in step (b) is between 40°

C. and 80°

C.

9. The method of claim 1, wherein said amino acid at position 4 of said amino acid sequence is mutated in comparison to said native sequence to an amino acid other than E, A, G, P, Q, or D.

10. A method for amplifying an RNA, that comprise:

(a) reverse transcribing said RNA according to a method of claim 1 to provide a cDNA;

(b) amplifying said cDNA.

11. A method of claim 10, wherein in step (b) said amplifying is carried out using a polymerase chain reaction.

12. A method for amplifying an RNA using a single-enzyme reverse transcription/amplification reaction, that comprises:

(a) providing an amplification reaction mixture comprising said RNA, a pair of primers, a divalent cation, and a mutant thermostable DNA polymerase, wherein said mutant DNA polymerase is characterized in that

i) in its native form said DNA polymerase comprises a polymerase domain comprising an amino acid sequence that is SEQ ID NO;

1, wherein said polymerase domain has the ability to incorporate nucleotides;

ii) the amino acid at position 2 of said amino acid sequence is S or A and the amino acid at position 5 of said amino acid sequence is L or I; and

iii) the amino acid at position 4 of said amino acid sequence is mutated in comparison to said native sequence to an amino acid other than E, A, G, or P; and

(b) treating said reaction mixture at a temperature sufficient for said mutant DNA polymerase to initiate synthesis of an extension product of said primer to provide a cDNA molecule complementary to said RNA;

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

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

13. The method of claim 12, wherein said mutant DNA polymerase in its native form comprises a polymerase domain comprising an amino acid sequence that is SEQ ID) NO:

2, the amino acid at position 3 of said amino acid sequence is Q or G, and the amino acid at position 6 of said amino acid sequence is S or A.

14. The method of claim 12, wherein said mutant DNA polymerase in its native form comprises a polymerase domain comprising an amino acid sequence that is SEQ ID NO:

3.

15. The method of claim 12, wherein said mutant DNA polymerase in its native form comprises a polymerase domain comprising an amino acid sequence that is SEQ ID NO:

4, and the amino acid at position 3 is Q or C.

16. The method of claim 12, wherein said mutant DNA polymerase is thermostable.

17. The method of claim 12, wherein said mutant DNA polymerase is a mutant form of a Thermus species DNA polymerase.

18. The method of claim 12, wherein said mutant DNA polymerase is a mutant form of Thermus thermophilus DNA polymerase or Thermus aquaticus DNA polymerase.

19. The method of claim 12, wherein said temperature of said reaction mixture in step(b) is between 40°

C. and 80°

C.

20. The method of claim 12, wherein said amino acid at position 4 of said amino acid sequence is mutated in comparison to said native sequence to an amino acid other than E, A, G, P, Q, or D.

21. A method for amplifying an RNA using a single-enzyme reverse transcription/amplification reaction, that comprises:

(a) providing an amplification reaction mixture comprising said RNA, a pair of primers, Mg+2, and a mutant thermostable DNA polymerase, wherein said mutant DNA polymerase is characterized in that

i) in its native from said DNA polymerase comprises a polymerase domain comprising an amino acid sequence that is SEQ ID NO;

1, wherein said polymerase domain has the ability to incorporate nucleotides;

ii) the amino acid at position 2 of said amino acid sequence is S or A and the amino acid at position 5 of said amino acid sequence is L or I; and

iii) the amino acid at position 4 of said amino acid sequence is mutated in comparison to said native sequence to an amino acid other than E, A, G, or P; and

(b) treating said reaction mixture at a temperature sufficient for said mutant DNA polymerase to initiate synthesis of an extension product of said primer to provide a cDNA molecule complementary to said RNA;

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

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

22. The method of claim 21, wherein said mutant DNA polymerase in its native form comprises an amino acid sequence that is SEQ ID) NO:

2, the amino acid at position 3 of said amino acid sequence is Q or G, and the amino acid at position 6 of said amino acid sequence is S or A.

23. The method of claim 21, wherein said mutant DNA polymerase in its native form comprises a polymerase domain comprising an amino acid sequence tat is SEQ ID NO:

3.

24. The method of claim 21, wherein said mutant DNA polymerase in its native form comprises an amino acid sequence that is SEQ ID NO:

4, and the amino acid at position 3 is Q or G.

25. The method of claim 21, wherein said mutant DNA polymerase is thermostable.

26. The method of claim 21, wherein said mutant DNA polymerase is a mutant form of a Thermus species DNA polymerase.

27. The method of claim 21, wherein said mutant DNA polymerase is a mutant form of Thermus thermophilus DNA polymerase or Thermus aquaticus DNA polymerase.

28. The method of claim 21, wherein said temperature of said reaction mixture in step (b) is between 40°

C. and 80°

C.

29. The method of claim 21, wherein said amino acid at position 4 of said amino acid sequence is mutated in comparison to said native sequence to an amino acid other than E, A, G, P, Q or D.