T4 DNA ligase variants with increased salt tolerance

A ligase, mutant technology, applied in the field of T4 DNA ligase variants, can solve the problems of sample loss, increase time and complexity, and achieve high activity effect

Active Publication Date: 2022-07-08
WUHAN AIBO TAIKE BIOTECH CO LTD
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Problems solved by technology

Such added purification steps add time and complexity to the experiment, and the purification procedure often results in sample loss - which can be significant

Method used

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  • T4 DNA ligase variants with increased salt tolerance
  • T4 DNA ligase variants with increased salt tolerance
  • T4 DNA ligase variants with increased salt tolerance

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Embodiment Construction

[0012] The term "biologically active fragment" refers to any fragment, derivative, homologue, or analog of a T4 DNA ligase mutant that has an in vivo or in vitro activity characteristic of a biomolecule; including, for example, ligase activity, or via a ligation repair band Mismatches present in nicked DNA. In some embodiments, the biologically active fragment, derivative, homologue or analog of the mutant T4 DNA ligase has any degree of biological activity of the mutant T4 DNA ligase in any in vivo or in vitro assay of interest .

[0013] In some embodiments, the biologically active fragment can optionally include any number of contiguous amino acid residues of a mutant T4 DNA ligase. The present invention also includes polynucleotides encoding any such biologically active fragments.

[0014] Biologically active fragments can result from post-transcriptional processing or translation of alternatively spliced ​​RNAs, or alternatively can be produced by engineering, batch syn...

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Abstract

A variety of T4DNA ligase mutants that exhibit enhanced ligation activity at high salt concentrations as compared to wild-type ligases are engineered, characterized and selected via gel electrophoresis of ligation products from standard ligation assays. A ligase catalyzes the formation of a phosphodiester bond between the 5'end and the 3 'end of complementary cohesive or blunt ends of double helix DNA, which process is crucial for many molecular biological processes, including cloning and sequencing.

Description

Background technique [0001] DNA ligases are indispensable repair proteins in living organisms that catalyze the repair of single-strand breaks in double-stranded DNA. T4 DNA ligase uses the energy of the biomolecule ATP to repair nicked double-stranded DNA, and also ligates double-stranded DNA with complementary single-stranded overhangs (sticky ends) as well as blunt-ended fragments, making it a useful tool for in vitro combinatorial smaller Useful tool for DNA fragments to generate double-stranded DNA constructs. [0002] DNA ligase forms phosphodiester bonds between duplex nucleic acid fragments at the intersection of juxtaposed 5' phosphate and 3' hydroxyl termini. By designing complementary overhangs between each double-stranded fragment, ligation can be both position-specific and directional. This allows the specific integration of DNA or RNA fragments into larger vectors to meet the needs of molecular biology research. Among commercially available ligases, T4 DNA lig...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/00C12N15/52C12N15/10
CPCC12N9/93C12Y605/01001C12N15/102C12Q2525/307C12Q2521/501C12Q2527/125C12Q1/6869
Inventor 朱振宇孙大鹏
Owner WUHAN AIBO TAIKE BIOTECH CO LTD
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