A novel simple and rapid PCR-based site-directed mutagenesis method |
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Authors: | Imen Rabhi Naouel Guedel Imen Chouk Khaled Zerria M Ridha Barbouche Koussay Dellagi Dahmani M Fathallah |
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Institution: | (1) Molecular Biotechnology Group, Laboratory of Immunology, Institut Pasteur de Tunis, BP 74, 1002 Bélvédère, Tunis, Tunisia |
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Abstract: | 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 α integrin
CD11b A-domain and the human CD8β cloned in pPICZαB, pGEX-2T, and CDM8 expression vectors, respectively. |
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Keywords: | Site-directed mutagenesis PCR primer recombinant protein structure function |
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