Site-Directed Mutagenesis Using Overlap Extension PCR

2003 ◽  
pp. 177-192 ◽  
Author(s):  
Ashok Aiyar ◽  
Yan Xiang ◽  
Jonathan Leis
Keyword(s):  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Overlap Extension Pcr ◽  
Overlap Extension

scholarly journals Efficient site-directed mutagenesis using an overlap extension-PCR method for expressing Mycoplasma hyopneumoniae genes in Escherichia coli

2009 ◽  
Vol 79 (1) ◽  
pp. 101-105 ◽  
Author(s):  
Simone Simionatto ◽  
Silvana B. Marchioro ◽  
Vanessa Galli ◽  
Tessália D. Luerce ◽  
Daiane D. Hartwig ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Mycoplasma Hyopneumoniae ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Overlap Extension Pcr ◽  
Overlap Extension ◽  
Pcr Method

scholarly journals Combined Overlap Extension PCR Method for Improved Site Directed Mutagenesis

2016 ◽  
Vol 2016 ◽  
pp. 1-7 ◽  
Author(s):  
Hasnain Hussain ◽  
Nikson Fatt-Ming Chong
Keyword(s):  
Codon Optimization ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Combined Method ◽  
Base Pairs ◽  
Single Experiment ◽  
Mutagenic Primer ◽  
Overlap Extension Pcr ◽  
Overlap Extension ◽  
Pcr Method

The combined overlap extension PCR (COE-PCR) method developed in this work combines the strengths of the overlap extension PCR (OE-PCR) method with the speed and ease of the asymmetrical overlap extension (AOE-PCR) method. This combined method allows up to 6 base pairs to be mutated at a time and requires a total of 40–45 PCR cycles. A total of eight mutagenesis experiments were successfully carried out, with each experiment mutating between two to six base pairs. Up to four adjacent codons were changed in a single experiment. This method is especially useful for codon optimization, where doublet or triplet rare codons can be changed using a single mutagenic primer set, in a single experiment.

Implications of Stisa2 catalytic residue restoration through site directed mutagenesis

2016 ◽  
Vol 42 (2) ◽  
Author(s):  
Hasnain Hussain ◽  
Nikson Fatt Ming Chong
Keyword(s):  
Catalytic Activity ◽  
Catalytic Triad ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Qualitative And Quantitative Analysis ◽  
Conserved Residues ◽  
Overlap Extension Pcr ◽  
Overlap Extension ◽  
Catalytic Network ◽  
And Function

AbstractObjective:Restoration of catalytic activity of Isa2 fromMethods:The six conserved amino acid residues absent in the Stisa2 gene were restored by mutation using the overlap extension PCR and the asymmetrical overlap extension PCR methods. Next, mutant Stisa2 with restored catalytic residues was expressed inResults:Both qualitative and quantitative analysis showed that the restoration of the conserved residues in the catalytic site did not restore starch debranching activity. Molecular modeling showed greater than expected distances between the catalytic triad in mutant Stisa2. These additional distances are likely to prevent hydrogen bonding which stabilizes the reaction intermediate, and are critical for catalytic activity.Conclusions:These results suggest that during evolution, mutations in other highly conserved regions have caused significant changes to the structure and function of the catalytic network. Catalytically inactive Isa2, which is conserved in starch-producing plants, has evolved important non-catalytic roles such as in substrate binding and in regulating isoamylase activity.

scholarly journals Multiple site-directed mutagenesis via simple cloning by prolonged overlap extension

BioTechniques ◽  
2020 ◽  
Vol 68 (6) ◽  
pp. 345-348
Author(s):  
Rasmus Hejlesen ◽  
Ernst-Martin Füchtbauer
Keyword(s):  
Dna Sequences ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Multiple Site ◽  
Overlap Extension

We describe the application of simple cloning by prolonged overlap extension for multiple site-directed mutagenesis in the same plasmid. We show that it is possible to use this technique with very short PCR templates. The technique is ideally suited for the generation of longer donor DNA sequences for CRISPR/Cas9-mediated homologous repair.

Site-directed mutagenesis by overlap extension using the polymerase chain reaction

Gene ◽  
1989 ◽  
Vol 77 (1) ◽  
pp. 51-59 ◽  
Author(s):  
Steffan N. Ho ◽  
Henry D. Hunt ◽  
Robert M. Horton ◽  
Jeffrey K. Pullen ◽  
Larry R. Pease
Keyword(s):  
Polymerase Chain Reaction ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Chain Reaction ◽  
Overlap Extension ◽  
Polymerase Chain

scholarly journals Site-directed mutagenesis in Brucella abortus S19 by overlap extension PCR-based procedure

2018 ◽  
Vol 68 (3) ◽  
pp. 273 ◽  
Author(s):  
B. NAYERI FASAEI ◽  
T. ZAHRAEI SALEHI ◽  
S. NASERLI ◽  
A. R SAEEDINIA ◽  
A. M. BEHROOZIKHAH
Keyword(s):  
Brucella Abortus ◽  
Genomic Structure ◽  
Bacterial Genome ◽  
Restriction Enzymes ◽  
Site Directed Mutagenesis ◽  
Economic Losses ◽  
Pcr Analysis ◽  
Modified Technique ◽  
Overlap Extension Pcr ◽  
Overlap Extension

Introduction of a site-directed mutation can be effective method to evaluate properties of various genes. Brucellosis is one of the most common zoonotic infectious diseases, which causes great economic losses. Thus, determination of pathogenicity factors in the genus Brucella can lead to the control of this health problem. Due to the importance of site-directed mutations in identification of genomic structure, overlap extension polymerase chain reaction (PCR) has been introduced as an improved technique for the removal and replacement of gene targets. In this study, three DNA fragments were amplified and combined using a two-step PCR with specific primers. The resulting fusion PCR product, obtained without any change in the nucleotide sequence, was cloned in a specific position in the pBluescript II SK (-) plasmid using restriction enzymes. Finally, the construct was transferred into cells of Brucella abortus S19 by electroporation and replaced the target gene (wbkA) in the genome of the bacterium. PCR analysis was performed on kanamycin-resistant colonies to provide genetic evidence that the B. abortus wbkA gene was interrupted by the kanamycin cassette. The results of this study show that the optimized modified technique, splicing by overlap extension PCR, is effective in creating mutations in the bacterial genome and can easily be used in Brucella spp.

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