Simultaneous Removal of Multiple DNA Segments by Polymerase Chain Reactions.
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.
Department of Biochemistry, School of Molecular and Cellular Biology, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, 314B Roger Adams Laboratory, Urbana, IL, 61801, USA.
Vishnu
V
Krishnamurthy
Department of Biochemistry, School of Molecular and Cellular Biology, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, 314B Roger Adams Laboratory, Urbana, IL, 61801, USA. kaizkaiz@illinois.edu.
Kai
K
Zhang
D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D005813:Genes, Synthetic; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction
D004247:DNA
Multiplex gene removal; Polymerase chain reaction; Restriction-free cloning; Synthetic DNA assembly and manipulation; Synthetic single-stranded bridging oligos