Multiplex gene removal by two-step polymerase chain reactions.
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.
Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
Vishnu Vardhan
VV
Krishnamurthy
Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
John S
JS
Khamo
Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
Ellen
E
Cho
Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
Cara
C
Schornak
Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA. Electronic address: kaizkaiz@illinois.edu.
Kai
K
Zhang
D000668:Ampicillin Resistance; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics*; D015723:Gene Library; D005796:Genes; D007613:Kanamycin Resistance; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*
D004247:DNA
Multiplex gene removal; PCR; Plasmids; Restriction-free cloning