A highly efficient procedure for site-specific mutagenesis of full-length plasmids using Vent DNA polymerase.
Careful titration of Vent polymerase activity allows efficient amplification of full-length plasmids (12 kb). The high processivity and fidelity of this enzyme made oligonucleotide-directed site-specific mutagenesis of plasmids a straight-forward process. Using only two primers, a mutagenic and a complementary, single-base mutants of recombinant plasmids were obtained consistently with > 90% efficiency from a single round of PCR. This procedure also made site-specific deletion, insertion, and several bases mutagenesis facile and efficient.
Byrappa
S
S
Rush-Presbyterian-St. Luke's Medical Center, Department of Immunology/Microbiology, Chicago, Illinois 60612, USA.
Gavin
D K
DK
Gupta
K C
KC
D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics*; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D018108:Respirovirus / Q000235:genetics
D017931:DNA Primers; C070331:Tli polymerase; D004259:DNA-Directed DNA Polymerase