Methods for preventing inhibition of nucleic acid synthesis by pyrophosphate

• US 6,764,839 B2
• Filed: 06/19/2001
• Issued: 07/20/2004
• Est. Priority Date: 11/22/1996
• Status: Expired due to Term

• Current Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Inventors: Blakesley, Robert W.
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): Kim, Young J.
• Application Number:
  US09/883,183
• Publication Number:
  US 20010055792A1
• Time in Patent Office: 1,127 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/183, 435/193, 435/195, 435/196, 536/23.1
• US Class Current: 435/91.1
• CPC Class Codes: C12N 9/1205 Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
• CPC Class Codes: C12N 9/1241 Nucleotidyltransferases (2.7.7)
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2527/127 the enzyme inhibitor or activator used
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)

Abstract

Methods for preventing inhibition of nucleic acid synthesis by pyrophosphate are disclosed. More specifically, the present invention concerns inhibiting or preventing pyrophosphorolysis in sequencing and amplification of nucleic acid molecules. According to the present invention, an enzyme which is a pentosyltransferase, a phosphotransferase with an alcohol group as acceptor, a nucleotidyltransferase, or a carboxy-lyase is added to the reaction which serves to remove pyrophosphate from the reaction mixture.

Claims

1. A method of making at least one nucleic acid molecule, said method comprising

(a) combining, in a mixture, at least one nucleic acid template, at least one polymerase or at least one reverse transcriptase, and an enzyme selected from the group consisting of a pentosyltransferase, a phosphotransferase with an alcohol group as acceptor, a nucleotidyltransferase, and a carboxy-lyase;

and

(b) incubating said mixture under conditions sufficient to make one or more first nucleic acid molecules complementary to all or a portion of said nucleic acid template.

2. The method of claim 1, wherein said enzyme is a pentosyltransferase.

3. The method of claim 2, wherein said enzyme is an adenine phosphoribosyltransferase or an orotate phosphoribosyltransferase.

4. The method of claim 1, wherein said enzyme is a phosphotransferase with an alcohol group as acceptor.

5. The method of claim 4, wherein said enzyme is a pyrophosphate:

glycerol phosphotransferase, a pyrophosphate;

serine phosphotransferase, a pyrophosphate;

fructose-6-phosphate 1-phosphotransferase or a pyrophosphate;

purine nucleoside kinase.

6. The method of claim 1, wherein said enzyme is a nucleotidyltransferase.

7. The method of claim 6, wherein said enzyme is an ATP:

sulfate adenylyltransferase, a UTP;

glucose-1-phosphate uridylyltransferase or an ATP;

glucose-1-phosphate adenylyltransferase.

8. The method of claim 1, wherein said enzyme is a carboxy-lyase.

9. The method of claim 8, wherein said enzyme is a phosphoenolpyruvate carboxykinase.

10. The method of claim 1, wherein said reverse transcriptase is a retroviral reverse transcriptase.

11. The method of claim 1, wherein said reverse transcriptase is an AMV reverse transcriptase or a RSV reverse transcriptase.

12. The method of claim 1, further comprising:

(c) incubating said one or more first nucleic acid molecules under conditions sufficient to synthesize one or more second nucleic acid molecules complementary to all or a portion of said one or more first nucleic acid molecules.

13. The method of claim 12, further comprising amplifying said first nucleic acid molecules or said second nucleic acid molecules.

14. The method of claim 1, wherein said polymerase is an RNA polymerase or a DNA polymerase.

15. The method of claim 14, wherein said DNA polymerase is thermostable.

16. A composition comprising:

(a) an enzyme selected from the group consisting of a pentosyltransferase, a phosphotransferase with an alcohol group as acceptor, a nucleotidyltransferase, and a carboxy-lyase; and

(b) at least one polymerase or at least one reverse transcriptase.

17. The composition of claim 16, wherein said enzyme of (a) is a pentosyltransferase.

18. The composition of claim 17, wherein said enzyme of (a) is an adenine phosphoribosyltransferase or an orotate phosphoribosyltransferase.

19. The composition of claim 16, wherein said enzyme of (a) is a phosphotransferase with an alcohol group as acceptor.

20. The composition of claim 19, wherein said enzyme of (a) is a pyrophosphate:

glycerol phosphotransferase, a pyrophosphate;

serine phosphotransferase, a pyrophosphate;

fructose-6-phosphate 1-phosphotransferase or a pyrophosphate;

purine nucleoside kinase.

21. The composition of claim 16, wherein said enzyme of (a) is a nucleotidyltransferase.

22. The composition of claim 21, wherein said enzyme is an ATP:

sulfate adenylyltransferase, a UTP;

glucose-1-phosphate uridylyltransferase or an ATP;

glucose-1-phosphate adenylyltransferase.

23. The composition of claim 16, wherein said enzyme of (a) is a carboxy-lyase.

24. The composition of claim 23, wherein said enzyme of (a) is a phosphoenolpyruvate carboxykinase.

25. The composition of claim 16, further comprising a substrate which is capable of either accepting a phosphate radical to give a phosphorylated product from pyrophosphate or effecting transfer of pyrophosphate when in the presence of said enzyme of (a).

26. The composition of claim 16, wherein said reverse transcriptase is a retroviral reverse transcriptase.

27. The composition of claim 16, wherein said reverse transcriptase is an AMV reverse transcriptase or a RSV reverse transcriptase.

28. The composition of claim 16, wherein said polymerase is an RNA polymerase or a DNA polymerase.

29. The composition of claim 28, wherein said DNA polymerase is thermostable.

30. A kit comprising an enzyme selected from the group consisting of a pentosyltransferase, a phosphotransferase with an alcohol group as acceptor, a nucleotidyltransferase, and a carboxy-lyase, a substrate which is capable of either accepting a phosphate radical to give a phosphorylated product from pyrophosphate or effecting transfer of pyrophosphate when in the presence of said enzyme;

and

at least one polymerase or at least one reverse transcriptase.

31. The kit of claim 30, wherein said reverse transcriptase is a retroviral reverse transcriptase.

32. The kit of claim 30, wherein said reverse transcriptase is an AMV reverse transcriptase or a RSV reverse transcriptase.

33. The kit of claim 30, wherein said polymerase is an RNA polymerase or a DNA polymerase.

34. The kit of claim 33, wherein said DNA polymerase is thermostable.