Simultaneous removal of multiple DNA segments by polymerase chain reactions

Vishnu Krishnamurthy, Kai Zhang

Research output: Chapter in Book/Report/Conference proceedingChapter


Precise DNA manipulation is a key enabling technology for synthetic biology. Approaches based on restriction digestion are often limited by the presence of certain restriction enzyme recognition sites. Recent development of restriction-free cloning approaches has greatly enhanced the flexibility and speed of molecular cloning. Most restriction-free cloning methods focus on DNA assembly. Much less work has been dedicated towards DNA removal. Here we introduce a protocol that allows simultaneous removal of multiple DNA segments from a plasmid using polymerase chain reactions (PCR). Our approach will be beneficial to applications in multiple sites mutagenesis, DNA library construction, genetic and protein engineering, and synthetic biology.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Number of pages11
StatePublished - Jan 1 2017

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745


  • Multiplex gene removal
  • Polymerase chain reaction
  • Restriction-free cloning
  • Synthetic DNA assembly and manipulation
  • Synthetic single-stranded bridging oligos

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics


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