Methods for repairing degraded DNA
First Claim
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1. A method for repairing and amplifying degraded DNA fragments in vitro comprising:
- I) contacting said degraded DNA fragments with a reaction mixture comprising;
a) a plurality of substantially purified enzymes, said enzymes having endonuclease activity, DNA N-glycosylase activity, AP lyase activity, polymerase activity, 3′
-diesterase activity, polynucleotide kinase activity, and DNA ligase activity, andb) a reaction buffer, wherein said reaction buffer further comprises ATP, wherein said contacting is under conditions such that said degraded DNA fragments are repaired, 5′
end phosphorylated, and ligated together to generate repaired concatamerized DNA;
II) purifying said repaired concatamerized DNA from said plurality of substantially purified enzymes to generate purified repaired concatamerized DNA; and
III) amplifying said purified repaired concatamerized DNA using a whole genome amplification method, wherein said whole genome amplification method employs random primers.
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Abstract
The present invention provides methods and kits for repair of degraded DNA which may then be used as a template for efficient amplification by a number of different amplification reactions. The method relies upon a series of enzymatic activities provided by DNA repair enzymes.
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Citations
34 Claims
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1. A method for repairing and amplifying degraded DNA fragments in vitro comprising:
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I) contacting said degraded DNA fragments with a reaction mixture comprising; a) a plurality of substantially purified enzymes, said enzymes having endonuclease activity, DNA N-glycosylase activity, AP lyase activity, polymerase activity, 3′
-diesterase activity, polynucleotide kinase activity, and DNA ligase activity, andb) a reaction buffer, wherein said reaction buffer further comprises ATP, wherein said contacting is under conditions such that said degraded DNA fragments are repaired, 5′
end phosphorylated, and ligated together to generate repaired concatamerized DNA;II) purifying said repaired concatamerized DNA from said plurality of substantially purified enzymes to generate purified repaired concatamerized DNA; and III) amplifying said purified repaired concatamerized DNA using a whole genome amplification method, wherein said whole genome amplification method employs random primers. - 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, 26)
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27. A method for repairing and amplifying degraded polynucleotide fragments comprising the steps of:
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a) providing a substantially purified polynucleotide kinase; b) providing a substantially purified polymerase; c) providing a substantially purified ligase to concatamerize polynucleotide fragments; d) providing one or more substantially purified enzymes that catalyze excision of degraded nucleotides from a polynucleotide; e) incubating a mixture of said degraded polynucleotide fragments with said polynucleotide kinase, said polymerase, said ligase, said one or more substantially purified enzymes, and ATP under conditions such that said degraded polynucleotide fragments are repaired, 5′
end phosphorylated, and ligated together to generate repaired concatamerized nucleic acid;f) purifying said repaired concatamerized nucleic acid from said polynucleotide kinase, said polymerase, said ligase, and said one or more substantially purified enzymes to generate purified repaired concatamerized nucleic acid; and g) amplifying said purified repaired concatamerized nucleic acid using a whole genome amplification method, wherein said whole genome amplification method employs random primers. - View Dependent Claims (28, 29, 30, 31, 32, 33, 34)
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Specification