Modified DNA-polymerase from carboxydothermus hydrogenoformans and its use for coupled reverse transcription and polymerase chain reaction

• US 20020160399A1
• Filed: 02/05/2002
• Published: 10/31/2002
• Est. Priority Date: 12/02/1997
• Status: Active Grant

• Current Assignee: Christine Ebenbichler, Gunthar Achhammer, Ursula Markau, Waltraud Ankenbauer
• Original Assignee: Christine Ebenbichler, Gunthar Achhammer, Ursula Markau, Waltraud Ankenbauer
• Inventors: Ebenbichler, Christine, Ankenbauer, Waltraud, Markau, Ursula, Achhammer, Gunthar
• Granted Patent: US 6,794,177 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Abstract

A purified DNA polymerase exhibiting reverse transcriptase activity in the presence of magnesium ions and/or manganese ions having reduced or no 5′-3′-exonuclease activity and substantially no RNaseH activity and obtainable from Carboxydothermus hydrogenoformans.

Claims

1. A purified modified DNA polymerase that is a modified form of a DNA polymerase obtainable from Carboxydothermus hydrogenoformans, wherein said modified DNA polymerase

(a) exhibits reverse transcriptase activity in the presence of magnesium ions and/or manganese ions;

(b) has reduced or no 5′

-3′

exonuclease activity; and

(c) has substantially no RNaseH activity.

2. A modified DNA polymerase of claim 1 that exhibits reverse transcriptase activity in the substantial absence of manganese ions.

3. A modified DNA polymerase of claim 2 having a molecular weight of about 64 to 71 kDa as determined by SDS polyacrylamide electrophoresis.

4. A modified DNA polymerase of claim 3 having amino acid sequence SEQ ID NO:

11.

20. A kit comprising an modified DNA polymerase of claim 1.

21. A kit comprising an modified DNA polymerase of claim 2.

22. A kit comprising an modified DNA polymerase of claim 3.

23. A kit comprising an modified DNA polymerase of claim 4.

5. A DNA sequence encoding a modified DNA polymerase that is a modified form of a DNA polymerase obtainable from Carboxydothermus hydrogenoformans, wherein said modified DNA polymerase

(a) exhibits reverse transcriptase activity in the presence of magnesium ions and/or manganese ions;

(b) has reduced or no 5′

-3′

exonuclease activity; and

(c) has substantially no RNaseH activity.

6. A DNA sequence of claim 5, wherein said modified DNA polymerase exhibits reverse transcriptase activity in the substantial absence of manganese ions.

7. A DNA sequence of claim 6, wherein said modified DNA polymerase has a molecular weight of about 64 to 71 kDa as determined by SDS polyacrylamide electrophoresis.

8. A DNA sequence of claim 7 encoding amino acid sequence SEQ ID NO:

11.

9. A DNA sequence of claim 8 having nucleotide sequence SEQ ID NO:

10.

10. A vector containing a DNA sequence of claim 5.

11. A vector containing a DNA sequence of claim 6.

12. A vector containing a DNA sequence of claim 7.

13. A vector containing a DNA sequence of claim 8.

14. A vector containing a DNA sequence of claim 9.

15. A vector of claim 14 that is pΔ

_2-225AR₄.

16. A host cell transformed with the vector of claim 15.

17. A host cell of claim 16 that is E.coli GA1.

18. A process for reverse transcribing RNA, comprising carrying out a reverse transcription reaction in a magnesium buffer using a modified form of a DNA polymerase obtainable from Carboxydothermus hydrogenoformans, wherein said modified DNA polymerase

(a) exhibits reverse transcriptase activity in the presence of magnesium ions and/or manganese ions;

(b) has reduced or no 5′

-3′

exonuclease activity; and

(c) has substantially no RNaseH activity.

19. A process of amplifying RNA, comprising carrying out a reverse transcription/polymerase chain reaction in a magnesium buffer using a combination of a first thermostable DNA polymerase and a second DNA polymerase, wherein said second DNA polymerase is a modified form of a DNA polymerase obtainable from Carboxydothermus hydrogenoformans, wherein said modified DNA polymerase

(a) exhibits reverse transcriptase activity in the presence of magnesium ions and/or manganese ions;

(b) has reduced or no 5′

-3′

exonuclease activity; and

(c) has substantially no RNaseH activity.