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Multiplex gene removal by two-step polymerase chain reactions

Author:
Krishnamurthy, Vishnu Vardhan   Khamo, John S.   Cho, Ellen   Schornak, Cara   Zhang, Kai  


Journal:
Analytical Biochemistry


Issue Date:
2015


Abstract(summary):

Abstract

Precise DNA manipulation is critical for molecular biotechnology. Restriction enzyme-based approaches are limited by their requirement of specific enzyme sites. Restriction-free cloning has greatly improved the flexibility and speed of precise DNA assembly. Most of these approaches focus on DNA assembly rather than gene removal. Here we present a polymerase chain reaction (PCR)-based cloning method that allows removal of multiple gene segments from plasmids without using restriction enzymes and thermostable ligase. We demonstrate simultaneous removal of three gene segments from a plasmid. This approach could be beneficial to DNA library construction, genetic and protein engineering, and synthetic biology.



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