Methods for preventing inhibition of nucleic acid synthesis by pyrophosphate

• US 7,344,835 B2
• Filed: 06/07/2004
• Issued: 03/18/2008
• Est. Priority Date: 11/22/1996
• Status: Expired due to Fees

• Current Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Inventors: Blakesley, Robert W.
• Primary Examiner(s): Kim; Young J.
• Application Number:
  US10/861,378
• Publication Number:
  US 20050239090A1
• Time in Patent Office: 1,380 Days
• Field of Search: None
• US Class Current: 435/6.11
• 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 reducing pyrophosphorolysis in a nucleic acid sequencing reaction, said method comprising incubating one or more deoxynucleotides, at least one dideoxynucleotide and a nucleic acid template together with an enzyme selected from the group consisting of a pentosyltransferase, a phosphotransferase with an alcohol group as acceptor, a nucleotidyltransferase, and a carboxy-lyase under conditions sufficient to form a second nucleic acid molecule complementary to all or a portion of said nucleic acid template.

2. The method of claim 1, further comprising bringing said enzyme into contact with a substrate that either accepts a phosphate radical to give a phosphorylated product from pyrophosphate, or that affects transfer of pyrophosphate when in the presence of said enzyme.

3. The method of claim 1, wherein said reaction comprises a thermostable DNA polymerase.

4. The method of claim 1, wherein said enzyme is phosphofructokinase from Thermoproteus tenax, phosphofructokinase from Bacillus stearothermophilus, uridylyltransferase from Methanococcus jannaschii, orotate phosphoribosyltransferase from Thermus thermophilus, or uracil phosphoribosyltransferase from Bacillus caldolyticus.

5. The method of claim 1, wherein said dideoxynucleotides are labeled.

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

7. The method of claim 1, wherein said dideoxynucleotide is ddATP.

8. The method of claim 1, wherein said dideoxynucleotide is ddTTP.

9. The method of claim 1, wherein said dideoxynucleotide is ddCTP.

10. The method of claim 1, wherein said dideoxynucleotide is ddGTP.