The patent badge is an abbreviated version of the USPTO patent document. The patent badge does contain a link to the full patent document.
The patent badge is an abbreviated version of the USPTO patent document. The patent badge covers the following: Patent number, Date patent was issued, Date patent was filed, Title of the patent, Applicant, Inventor, Assignee, Attorney firm, Primary examiner, Assistant examiner, CPCs, and Abstract. The patent badge does contain a link to the full patent document (in Adobe Acrobat format, aka pdf). To download or print any patent click here.
Patent No.:
Date of Patent:
Oct. 06, 2020
Filed:
Nov. 09, 2016
Zhen Kang, Wuxi, CN;
Jian Chen, Wuxi, CN;
Peng Jin, Wuxi, CN;
Guocheng Du, Wuxi, CN;
Wenwen Ding, Wuxi, CN;
Zhen Kang, Wuxi, CN;
Jian Chen, Wuxi, CN;
Peng Jin, Wuxi, CN;
Guocheng Du, Wuxi, CN;
Wenwen Ding, Wuxi, CN;
Jiangnan University, Wuxi, JS, CN;
Abstract
The present invention provides a method for scarless in vitro DNA assembly using thermostable exonucleases and ligase, which relates to the field of genetic engineering. The present invention provides a fast method for assembling DNA subfragments with homologous ends, which employs thermostable polymerases and ligase in a thermal cycle of denaturation, annealing, digestion and ligation. After denaturation, DNA subfragments are assembled together via annealing of the homologous end sequences, the unpaired single-stranded overhangs are digested by polymerases, and the resulting nicked gaps are sealed by a ligase. Using this method, 2-6 DNA subfragments were successfully assembled within two hours. This method can be used in conventional DNA recombination and be adapted to high throughput assembly operations. In addition, combinatorial mutations can be easily introduced into the assembled sequence by use of primers with mutated bases. It is particularly suitable for making enzyme and synthetic pathways mutation libraries with high diversity, which can be used in directed evolution to screen for enzymes and synthetic pathways with desirable properties.