1. Multiplex gene removal by two-step polymerase chain reactions.
- Author
-
Krishnamurthy VV, Khamo JS, Cho E, Schornak C, and Zhang K
- Subjects
- Ampicillin Resistance, Base Sequence, Gene Library, Genes, Kanamycin Resistance, Cloning, Molecular methods, DNA genetics, Plasmids genetics, Polymerase Chain Reaction methods
- 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., (Copyright © 2015 Elsevier Inc. All rights reserved.)
- Published
- 2015
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