Methods for preventing inhibition of nucleic acid synthesis by pyrophosphate
First Claim
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.
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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.
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Citations
34 Claims
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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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
(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.
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13. The method of claim 12, further comprising amplifying said first nucleic acid molecules or said second nucleic acid molecules.
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14. The method of claim 1, wherein said polymerase is an RNA polymerase or a DNA polymerase.
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15. The method of claim 14, wherein said DNA polymerase is thermostable.
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16. A composition comprising:
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(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. - View Dependent Claims (17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29)
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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;
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at least one polymerase or at least one reverse transcriptase. - View Dependent Claims (31, 32, 33, 34)
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Specification