A novel simple and rapid PCR-based site-directed mutagenesis method.
Site-directed mutagenesis (SDM) is a powerful tool for exploring protein structure and function, and several procedures adjusted to specific purposes are still being developed. Herein we describe a straightforward and efficient method with versatile applications for introducing site-specific alterations in any deoxyribonucleic acid (DNA) sequence cloned in a plasmidic expression vector. In this polymerase chain reaction (PCR)-based SDM method, forward and reverse primers are used to amplify the plasmid containing the sequence of interest. The primers are designed so that the desired modifications are introduced at the 5' end of one of the primers, whereas the other primer starts with the nucleotide at position (-1) of the one to be modified. The PCR is carried out using Pfu DNA polymerase. The blunt-ended PCR-generated DNA fragment is self-ligated and used to transform Escherichia coli. Mutant clones are screened by colony hybridization using the mutagenic primer as probe and the presence of the mutation is confirmed by direct DNA sequencing. This procedure was used efficiently to introduce substitutions, deletions, and insertions in the DNA sequences coding for a recombinant form (scFv) of antibody 107 specific of the human CR3 molecule, the rat alpha integrin CD11b A-domain and the human CD8beta cloned in pPICZalphaB, pGEX-2T, and CDM8 expression vectors, respectively.
Rabhi
Imen
I
Molecular Biotechnology Group, Laboratory of Immunology, Institut Pasteur de Tunis, BP 74, 1002 Bélvédère, Tunis, Tunisia.
Guedel
Naouel
N
Chouk
Imen
I
Zerria
Khaled
K
Barbouche
M Ridha
MR
Dellagi
Koussay
K
Fathallah
Dahmani M
DM
D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D017353:Gene Deletion; D005821:Genetic Techniques; D005822:Genetic Vectors; D006801:Humans; D017403:In Situ Hybridization; D008274:Magnesium / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009693:Nucleic Acid Hybridization; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D011994:Recombinant Proteins; D004247:DNA; C072146:Pfu DNA polymerase; D004259:DNA-Directed DNA Polymerase; D008274:Magnesium