Multiple ligase compositions, systems, and methods
AssigneeRgene, Inc.
InventorsYu Zheng, Manqing Hong
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Abstract
Provided herein are compositions, systems, and methods using multiple ligases, wherein at least one of the ligases is an adenylation-deficient ATP-dependent ligase or an un-adenylated ATP-dependent ligase (e.g., present in an ATP free mixture). In certain embodiments, multiple ligases are used to ligate a pre-adenylated double stranded sequence to a non-adenylated double stranded sequence (e.g., the adenylation-deficient ATP-dependent ligase or un-adenylated ATP-dependent ligase ligates the first strand, and a second ligase ligates the second strand). In other embodiments, provided herein are mutant T4 ligases (e.g., K159S mutant or K159C mutant).
Claims3
A method of ligating a nucleic acid sequence comprising:
- (a) combining the following components into a reaction mixture:
- (i) a first ligase, wherein the first ligase is a T4 DNA ligase mutant comprising the amino acid sequence of SEQ ID NO:
- 1 or the amino acid sequence of SEQ ID NO:
- 2;
- (ii) an adenylated double-stranded nucleic acid sequence (ADSNAS);
- and
- (iii) a non-adenylated double stranded nucleic acid sequence (non-ADSNAS);
- wherein the non-ADSNAS comprises a first strand hybridized to a second strand, and wherein combining the components is under conditions such that the first ligase ligates the first strand of the non-ADSNAS to the ADSNAS;
- and
- (b) adding a second ligase to the reaction mixture under conditions such that the second ligase ligates the second strand of the non-ADSNAS to the ADSNAS, wherein the second ligase is an ATP-dependent ligase or a NAD-dependent ligase.
Dependencies are inferred from the claim text.