Repair of Nucleic Acids for Improved Amplification
First Claim
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1. A method for repairing a damaged polynucleotide so as to enhance at least one of fidelity and yield of a copied or amplified product of the polynucleotide, comprising:
- (a) incubating the polynucleotide in a reaction mixture comprising;
an effective amount of at least one apurinic/apyrimidinic (AP) endonuclease;
a DNA ligase; and
at least one of NAD+ or ATP as a cofactor; and
(b) enhancing at least one of fidelity and yield of the copied or amplified product.
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Abstract
Methods and compositions are provided for repairing a polynucleotide so that it can be copied with improved fidelity and/o yield in, for example, an amplification reaction. This involves the use of a reaction mixture that includes a DNA ligase and an effective amount of at least one endonuclease as well as a cofactor selected from NAD+ or ATP.
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Citations
56 Claims
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1. A method for repairing a damaged polynucleotide so as to enhance at least one of fidelity and yield of a copied or amplified product of the polynucleotide, comprising:
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(a) incubating the polynucleotide in a reaction mixture comprising;
an effective amount of at least one apurinic/apyrimidinic (AP) endonuclease;
a DNA ligase; and
at least one of NAD+ or ATP as a cofactor; and(b) enhancing at least one of fidelity and yield of the copied or amplified product. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25)
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26. A kit, comprising:
- two or more enzymes for forming a reaction mixture wherein at least one of the enzymes is a DNA ligase, and at least one of the enzymes is an AP endonuclease at a concentration in the range of 0.0001 units/μ
l to 100 units/μ
l in the reaction mixture, the two or more enzymes being formulated for addition to a damaged polynucleotide preparation to enhance repair of the polynucleotide; and
instructions for its use.
- two or more enzymes for forming a reaction mixture wherein at least one of the enzymes is a DNA ligase, and at least one of the enzymes is an AP endonuclease at a concentration in the range of 0.0001 units/μ
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27. A polynucleotide repair mixture, comprising:
- an effective amount of at least one AP endonuclease, a DNA ligase, and a DNA polymerase, wherein the repair mixture can be added to a polynucleotide and the polynucleotide can be amplified without removing or degrading the polynucleotide repair mixture; and
wherein the repair mixture enhances at least one of yield and fidelity of a copied polynucleotide. - View Dependent Claims (28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 49, 50, 51, 52, 53, 54, 55, 56)
- an effective amount of at least one AP endonuclease, a DNA ligase, and a DNA polymerase, wherein the repair mixture can be added to a polynucleotide and the polynucleotide can be amplified without removing or degrading the polynucleotide repair mixture; and
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47. A polynucleotide repair mixture, comprising:
- Bst DNA polymerase, AP endonuclease, endonuclease VIII, a DNA ligase, Fpg, PDG and UDG.
- View Dependent Claims (48)
Specification