Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1997-11-17
2001-09-18
Houtteman, Scott W. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S196000
Reexamination Certificate
active
06291164
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to methods for nucleic acid synthesis. Specifically, the present invention relates to DNA synthesis via a primer extension reaction and methods for RNA synthesis. In particular, the invention relates to methods for avoiding the inhibiting effects of pyrophosphate on RNA synthesis and primer extension DNA reactions, for example, polymerase chain reactions (PCRs) and sequencing reactions.
2. Background of the Invention
It has been recognized that pyrophosphorolysis, where an oligonucleotide is reduced in length, is detrimental to primer extension reactions. The pyrophosphorolysis is caused by the availability of pyrophosphate. For example, PCR is inhibited by the addition of pyrophosphate even at very low concentrations. According to U.S. Pat. No. 5,498,523, this pyrophosphorolysis can be prevented by providing an agent, for example, a pyrophosphatase, capable of removing pyrophosphate. Addition of pyrophosphatase to a PCR greatly enhances the progress of the reaction and provides superior results compared to the reaction without a pyrophosphatase. See U.S. Pat. No. 4,800,159 more uniformity in intensities of bands formed in a polyacrylamide gel used to identify products of the sequencing reaction. This uniformity is due to prevention of degradation of specific DNA products by pyrophosphorolysis. See also, Tabor, S. and Richardson, C. C.,
J. Biol. Chem
. 265:8322 (1990); U.S. Pat. No. 4,962,020; and Ruan, C. C. et al.,
Comments
17(1):1 (1990).
Each product or band in a dideoxy sequencing experiment is a polynucleotide complementary to the template and terminated at the 3′ end in a base-specific manner with a dideoxynucleotide. The dideoxy stabilizes the product, preventing further polymerization of the polynucleotide. However, in certain regions of the template, the bands, especially after prolonged reaction, will reduce in intensity or completely disappear (“drop-out” bands). A drop-out may not be readily detected by the operator, leading to errors in the interpretation of the data either by a human or computer-driven analyzer. Since this phenomenon is stimulated by inorganic pyrophosphate, the effect is presumably due to pyrophosphorolysis (reverse polymerization), not 3-exonucleolytic activity. It is hypothesized that DNA polymerase idling at the end of these terminated products and in the presence of sufficient pyrophosphate will remove the dideoxynucleotide, then extend from the now free 3′-hydroxyl end to another dideoxy termination. In effect, the bands are converted to longer polynucleotides/bands. Removal of pyrophosphate as it is generated in the polymerization reaction eliminates this problem.
Researchers have used a series of enzyme reactions coupled to pyrophosphate generation to measure DNA polymerase activity. In the first (P. Nyren,
Anal. Biochem
. 167:235 (1987)), Nyren used ATP: sulfate adenylyl-transferase to convert pyrophosphate and adenosine 5′-phosphosulfate to ATP and sulfate ion. The ATP was used to make light with luciferase. In the second (J. C. Johnson et al.,
Anal. Biochem
. 26:137 (1968)), the researchers reacted the pyrophosphate with UDP-glucose in the presence of UTP: glucose-1-phosphate uridylyltransferase to produce UTP and glucose-1-phosphate. In two more steps, polymerase activity was measured spectrophotometrically by the conversion of NADP to NADPH. While these articles describe the use of ATP: sulfate adenylyltransferase and UTP: glucose-1-phosphate uridylyltransferase in measuring DNA polymerase activity, they do not describe their use to prevent or inhibit pyrophosphorolysis in nucleic acid synthesis reactions.
SUMMARY OF THE INVENTION
A number of naturally-occurring enzymes use pyrophosphate as a substrate, including certain transferases, kinases and lyases. By coupling the reaction catalyzed by one of these enzymes to the polymerase reaction, pyrophosphate will not build up, preventing pyrophosphorolysis in nucleic acid synthesis reactions. Thus, the present invention relates to a method of inhibiting or preventing pyrophosphorolysis during synthesis of a nucleic acid molecule, said method comprising
(a) combining one or more nucleotides and a nucleic acid template;
(b) incubating the one or more nucleotides and nucleic acid template together with a polymerase and an enzyme selected from the group consisting of a pentosyltransferase, a phosphotransferase with 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 the nucleic acid template.
The method of the invention more specifically relates to a method of inhibiting or preventing pyrophosphorolysis, said method comprising
(a) combining a primer with a nucleic acid template under conditions sufficient to form a hybridized product; and
(b) incubating said hybridized product in the presence of (i) one or more nucleotides, (ii) a polymerase, and (iii) an enzyme selected from the group consisting of a pentosyltransferase, a phosphotransferase with alcohol group as acceptor, a nucleotidyltransferase, and a carboxy-lyase under conditions sufficient to synthesize a second nucleic acid molecule complementary to all or a portion of said nucleic acid template.
Specifically, the method of the present invention relates to inhibition of pyrophosphorolysis in the synthesis of DNA and RNA molecules using the appropriate nucleotides and polymerases (dNTP's/rNTP's and DNA polymerase/RNA polymerase).
In particular, the present invention may be used in primer extension reactions to prevent the inhibition of nucleic acid synthesis during amplification and may be used to prevent band drop out in sequencing reactions. Thus, the invention relates to a method to prevent inhibition of nucleic acid synthesis during amplification of a double stranded nucleic acid molecule comprising
(a) providing a first and second primer, wherein said first primer is complementary to a sequence at or near the 3′ termini of the first strand of said nucleic acid molecule and said second primer is complementary to a sequence at or near the 3′ termini of the second strand of said nucleic acid molecule;
(b) hybridizing said first primer to said first strand and said second primer to said second strand in the presence of (i) a polymerase, and (ii) an enzyme selected from the group consisting of a pentosyltransferase, a phosphotransferase with an alcohol group as an acceptor, a nucleotidyltransferase and a carboxy-lyase under conditions such that a third nucleic acid molecule complementary to said first strand and a fourth nucleic acid molecule complementary to said second strand are synthesized;
(c) denaturing said first and third strand and said second and fourth strand; and
(d) repeating steps (a) to (c) one or more times.
The present invention also relates to a method of sequencing a DNA molecule comprising:
(a) combining a primer with a first DNA molecule under conditions sufficient to form a hybridized product;
(b) contacting said hybridized product with nucleotides, a DNA polymerase, 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 a terminator nucleotide to give a reaction mixture;
(c) incubating the reaction mixture under conditions sufficient to synthesize a random population of DNA molecules complementary to said first DNA molecule, wherein said synthesized DNA molecules are shorter in length than said first DNA molecule and wherein said synthesized DNA molecules comprise a terminator nucleotide at their 3′ termini; and
(d) separating said synthesized DNA molecules by size so that at least a part of the nucleotide sequence of said first DNA molecule can be determined.
The invention also relates to a solution for use in nucleic acid synthesis, amplification or sequencing, comprising
(a) an enzyme selected from the group consisting of
Houtteman Scott W.
Invitrogen Corporation
Sterne Kessler Goldstein & Fox P.L.L.C.
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