Compositions and Methods for Enhanced Sensitivity and Specificity of Nucleic Acid Synthesis
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Abstract
The present invention relates to polypeptides, compositions and methods for enhancing synthesis of nucleic acid molecules. In a preferred aspect, the invention relates to inhibition or control of nucleic acid synthesis, sequencing or amplification. Specifically, the present invention discloses polypeptides having affinity for double-stranded and/or single-stranded nucleic acid molecules and/or single-stranded/double-stranded nucleic acid complexes (e.g., primer/template complexes, double-stranded templates, single-stranded templates or single-stranded primers) for use in such enhanced synthesis and more particularly to polymerases having reduced polymerase and optionally reduced exonuclease activities (3′ to 5′ and/or 5′ to 3′ exonuclease activity), and to nucleases having reduced nuclease activity. The polypeptides of the invention are capable of inhibiting nonspecific nucleic acid synthesis at ambient temperature. Thus, in a preferred aspect, the invention relates to “hot start” synthesis of nucleic acid molecules. Accordingly, the invention prevents non-specific nucleic acid synthesis at low temperatures, for example during reaction set up. The invention also relates to kits for synthesizing, amplifying, reverse transcribing or sequencing nucleic acid molecules comprising one or more of the polypeptides or compositions of the invention. The invention also relates to compositions prepared for carrying out the methods of the invention and to compositions made after or during such methods. The invention also generally relates to polypeptides and compositions useful for inhibiting or preventing degradation of various nucleic acid molecules.
52 Citations
59 Claims
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1-56. -56. (canceled)
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57. A method for amplifying double-stranded DNA molecule, comprising:
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(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 DNA molecule and said second primer is complementary to a sequence at or near the 3′
-termini of the second strand of said DNA molecule and art inhibitory polypeptide which has been mutated to substantially reduce or eliminate polymerase and/or exonuclease activity in said polypeptide, under conditions such that said polypeptide prevents or inhibits nucleic(b) hybridizing said first primer to said first strand and said second primer to said second strand to form hybridized molecules;
(c) incubating said hybridized molecules under conditions sufficient to inactivate or denature said polypeptide and sufficient to allow synthesis of a third DNA molecule complementary to said first strand and a fourth DNA molecule complementary to said second strand;
(d) denaturing said first and third strand, and said second and fourth strands; and
(e) repeating steps (a) to (c) or (a) to (d) one or more times.
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58. A method of preparing cDNA from mRNA, comprising
(a) forming a mixture by mixing one or more mRNA templates with an inhibitory polypeptide which has been mutated to substantially reduce or eliminate polymerase and/or exonuclease activity in said polypeptide; - and
(b) incubating said mixture under conditions sufficient to synthesize a cDNA molecule complementary to all or a portion of said one or more templates, wherein said cDNA molecule is synthesized.
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59. A method for amplifying a nucleic acid molecule comprising:
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(a) forming a mixture by mixing one or more nucleic acid templates with an inhibitory polypeptide which has been mutated to substantially reduce or eliminate polymerase and/or exonuclease activity in said polypeptide under conditions sufficient to prevent or inhibit nucleic acid amplification; and
(b) incubating said mixture under conditions sufficient to inactivate or denature said polypeptide and sufficient to allow synthesis of a nucleic acid molecule complementary to all or a portion of said one or more templates, wherein said nucleic acid molecule is amplified.
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Specification