Multiplex gene removal by two-step polymerase chain reactions

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

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish (US)
Pages (from-to)7-9
Number of pages3
JournalAnalytical Biochemistry
Volume481
DOIs
StatePublished - Jun 3 2015

Keywords

  • Multiplex gene removal
  • PCR
  • Plasmids
  • Restriction-free cloning

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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