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US 10,301,660 B2Granted
Methods and compositions for repair of DNA ends by multiple enzymatic activities
AssigneeTakara Bio USA, Inc.
InventorsEmmanuel Kamberov, John Langmore, Tim Tesmer, Marta Gonzalez-Plasky
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Abstract
Provided herein are compositions for and methods of generating ligation-competent nucleic acids. In some aspects, the compositions comprise Exonuclease III, T4 DNA Polymerase, Klenow, and/or T4 polynucleotide kinase.
Claims36
A method of generating a ligation-competent nucleic acid molecule, the method comprising:
- obtaining a composition comprising T4 DNA polymerase, T4 polynucleotide kinase, exonuclease III, and Klenow;
- and
- incubating the composition with a nucleic acid in need of end repair under conditions to allow for the end repair of the nucleic acid molecule such that enzymatic activities of the T4 DNA polymerase, T4 polynucleotide kinase, exonuclease III, and Klenow generate a nucleic acid product having blunt ends with a 5′-phosphate group and a 3′-hydroxyl group,
- thereby producing a ligation-competent nucleic acid molecule.
1 more element
A method of generating a ligation-competent nucleic acid molecule, the method comprising:
- obtaining a nucleic acid molecule in need of end repair;
- adding at least T4 DNA polymerase, T4 polynucleotide kinase, and exonuclease III to the nucleic acid molecule;
- and
- incubating the nucleic acid molecule with the at least T4 DNA polymerase, T4 polynucleotide kinase, and exonuclease III under conditions to allow for the end repair of the nucleic acid molecule such that enzymatic activities of the T4 DNA polymerase, T4 polynucleotide kinase, and exonuclease III generate a nucleic acid product having blunt ends with a 5′-phosphate group and a 3′-hydroxyl group,
- thereby producing a ligation-competent nucleic acid molecule.
2 more elements
A method of preparing a library of ligation-competent DNA fragments, the method comprising:
- a) providing a composition comprising a plurality of DNA fragments in need of end repair to be ligated;
- b) adding T4 DNA polymerase, T4 polynucleotide kinase, and exonuclease III to the composition;
- and
- c) incubating the composition under conditions to allow for the end repair of the plurality of DNA fragments such that enzymatic activities of the T4 DNA polymerase, T4 polynucleotide kinase, and exonuclease III generate a plurality of ligation-competent DNA fragments, wherein each DNA fragment has blunt ends with a 5′-phosphate group and a 3′-hydroxyl group, thereby producing a library of ligation-competent DNA fragments.
1 more element
Dependencies are inferred from the claim text.