TY - JOUR
T1 - A fast and simple method for simultaneous mixed site-specific mutagenesis of a wide coding sequence
AU - Follo, Carlo
AU - Isidoro, Ciro
PY - 2008/2
Y1 - 2008/2
N2 - Background site-specific mutagenesis at one or multiple sites has recently become an invaluable strategy in functional proteomic studies and genetic engineering. In the present paper we describe a novel PCR-based procedure for site-specific mutagenesis that permits, in a single-step, all three types of nucleotide sequence mutation (deletion, insertion and substitution). The entire procedure is carried out in one tube and takes about 3-4 h. The method utilizes two primers, one of which is phosphorylated at the 5′-terminus, that are designed to directly anneal back-to-back to the target sequence inserted in a plasmid. For the deletion type of mutagenesis (which has virtually no limit to its extent), primers anneal at the ends of the sequence to be deleted. For insertion and substitution types of mutagenesis the primers bear the mutagenic sequences in a tail. The entire circular plasmid, here tested for a maximum length of 7 kbp, is amplified by inverse PCR. The PCR product incorporates the desired mutagenesis and, after ligation, the plasmid is ready for cloning into bacteria. The method has been proved very efficient for deletions of up to 279 nucleotides, for introducing simultaneous deletions, insertions and substitutions, and for performing alanine scanning over a wide coding region. The procedure is suitable for applications in genetic engineering and for the construction of libraries.
AB - Background site-specific mutagenesis at one or multiple sites has recently become an invaluable strategy in functional proteomic studies and genetic engineering. In the present paper we describe a novel PCR-based procedure for site-specific mutagenesis that permits, in a single-step, all three types of nucleotide sequence mutation (deletion, insertion and substitution). The entire procedure is carried out in one tube and takes about 3-4 h. The method utilizes two primers, one of which is phosphorylated at the 5′-terminus, that are designed to directly anneal back-to-back to the target sequence inserted in a plasmid. For the deletion type of mutagenesis (which has virtually no limit to its extent), primers anneal at the ends of the sequence to be deleted. For insertion and substitution types of mutagenesis the primers bear the mutagenic sequences in a tail. The entire circular plasmid, here tested for a maximum length of 7 kbp, is amplified by inverse PCR. The PCR product incorporates the desired mutagenesis and, after ligation, the plasmid is ready for cloning into bacteria. The method has been proved very efficient for deletions of up to 279 nucleotides, for introducing simultaneous deletions, insertions and substitutions, and for performing alanine scanning over a wide coding region. The procedure is suitable for applications in genetic engineering and for the construction of libraries.
KW - Alanine scanning
KW - Cathepsin D
KW - Mixed site-specific mutagenesis
KW - Nucleotide sequence mutation
KW - Wide coding sequence
UR - http://www.scopus.com/inward/record.url?scp=38749113960&partnerID=8YFLogxK
U2 - 10.1042/BA20070045
DO - 10.1042/BA20070045
M3 - Article
SN - 0885-4513
VL - 49
SP - 175
EP - 183
JO - Biotechnology and Applied Biochemistry
JF - Biotechnology and Applied Biochemistry
IS - 2
ER -