Overlap Extension PCR Cloning

2011 ◽  
Vol 1 ◽  
Author(s):  
Mary Johnson
Keyword(s):  
Pcr Cloning ◽  
Overlap Extension Pcr ◽  
Overlap Extension

Overlap Extension PCR Cloning

2013 ◽  
pp. 31-42 ◽  
Author(s):  
Anton Bryksin ◽  
Ichiro Matsumura
Keyword(s):  
Pcr Cloning ◽  
Overlap Extension Pcr ◽  
Overlap Extension

scholarly journals Overlap extension PCR cloning: a simple and reliable way to create recombinant plasmids

BioTechniques ◽  
2010 ◽  
Vol 48 (6) ◽  
pp. 463-465 ◽  
Author(s):  
Anton Bryksin ◽  
Ichiro Matsumura
Keyword(s):  
Pcr Cloning ◽  
Recombinant Plasmids ◽  
Overlap Extension Pcr ◽  
Overlap Extension

Bioinformatic Analysis in Designing Mega-primer in Overlap Extension PCR Cloning (OEPC) Technique

2021 ◽  
Vol 5 (2) ◽  
Author(s):  
Mardalisa ◽  
Sony Suhandono ◽  
Novi Yanti ◽  
Fazrol Rozi ◽  
Fitri Nova ◽  
...  
Keyword(s):  
Dna Analysis ◽  
Bioinformatic Analysis ◽  
Amino Acid Sequences ◽  
Homologous Sequence ◽  
Blue Color ◽  
Success Rates ◽  
Pcr Cloning ◽  
Overlap Extension Pcr ◽  
Complex Processes ◽  
Overlap Extension

Bioinformatics has developed into an application tool for basic and applied research in the biomedical and biotechnology field. Polymerase Chain Reaction (PCR) is a common technique in the molecular area that has always involved bioinformatics science. PCR cloning techniques such as TA cloning and PCR-mediated cloning exhibit complex processes with low success rates. One easy, effective, and practical solution is to use a mega-primer with the Overlap Extension PCR Cloning (OEPC) technique. The success of PCR cloning using the mega-primer design in the OEPC technique is strongly influenced by the characteristics of the mega-primer used. Knowledge of mega-primer characteristics is one of the important factors in the success of PCR cloning. The design process for the mega-primer str promoter was characterized based on the principle of a genetic algorithm using the web-based bioinformatics tools such as ClustalW, NetPrimer, and BLAST. The success of the mega-primer construction in producing recombinant pSB1C3 vector has been confirmed by the sequencing method and the function of the reporting protein (AmilCP). DNA analysis shows a 100 % homologous sequence on the str promoter, while  E. coli colonies successfully express the purplish-blue color. Mega-primer characters can save costs and time of the research by maintaining the primer parameters that provide optimal values and increase the success value of PCR cloning via bioinformatics software. Hence, implications on biological problems, especially using DNA and amino acid sequences, could solve rapidly.

scholarly journals Aptamer-dependent full-length cDNA synthesis by overlap extension PCR

BioTechniques ◽  
2004 ◽  
Vol 37 (1) ◽  
pp. 124-129 ◽  
Author(s):  
Yasumasa Mitani ◽  
Takayuki Nakayama ◽  
Matthias Harbers ◽  
Yoshihide Hayashizaki
Keyword(s):  
Full Length ◽  
Cdna Synthesis ◽  
Full Length Cdna ◽  
Overlap Extension Pcr ◽  
Overlap Extension

A Combination Strategy for Construction of Peptide-��2m-H-2Kb Single Chain with Overlap Extension PCR and One-Step Cloning

2016 ◽  
Vol 26 (12) ◽  
pp. 2184-2191 ◽  
Author(s):  
Tao Xu ◽  
Xiaoe Li ◽  
You Wu ◽  
Khawar Ali Shahzad ◽  
Wei Wang ◽  
...  
Keyword(s):  
Single Chain ◽  
Combination Strategy ◽  
Overlap Extension Pcr ◽  
Overlap Extension ◽  
One Step

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 A Short Protocol for Gene Knockout and Complementation in Xylella fastidiosa Shows that One of the Type IV Pilin Paralogs (PD1926) Is Needed for Twitching while Another (PD1924) Affects Pilus Number and Location

2018 ◽  
Vol 84 (18) ◽  
Author(s):  
Prem P. Kandel ◽  
Hongyu Chen ◽  
Leonardo De La Fuente
Keyword(s):  
Natural Transformation ◽  
Gene Knockout ◽  
Xylella Fastidiosa ◽  
Twitching Motility ◽  
Wild Type ◽  
Content Type ◽  
Overlap Extension Pcr ◽  
Type Iv ◽  
Overlap Extension ◽  
Pilin Subunit

ABSTRACT Twitching motility is one of the major virulence factors of the plant-pathogenic bacterium Xylella fastidiosa, and it is mediated by type IV pili (TFP) that are present at one of the cell poles. Genome analysis of X. fastidiosa showed the presence of at least four paralogs of the gene pilA, which encodes the TFP major pilin subunit. However, whether all of these paralogs have a functional role in TFP structure and function is unknown. Here, using a short and reliable protocol based on overlap extension PCR and natural transformation, deletion mutants of two pilA paralogs (pilA1 PD1924 and pilA2 PD1926) were generated in two X. fastidiosa subsp. fastidiosa strains, WM1-1 and TemeculaL, followed by assessment of twitching motility and biofilm formation. Deletion of pilA2 caused loss of twitching motility, whereas deletion of pilA1 did not influence twitching motility but caused hyperpiliation and extended distribution of TFP along the sides of the cell. Loss of twitching motility due to pilA2 deletion was restored when a wild-type copy of the pilA2 gene was added at a neutral site in the genome of mutants in both wild-type backgrounds. This study demonstrates that PCR templates generated by overlap extension PCR can be successfully used to rapidly generate gene knockouts and perform genetic complementation in X. fastidiosa, and that twitching motility in X. fastidiosa is controlled by regulating the transcription of the major pilin subunit, pilA2. IMPORTANCE The bacterial plant pathogen Xylella fastidiosa causes incurable diseases in multiple hosts, including grape, citrus, and blueberry. Historically restricted to the Americas, it was recently found to cause epidemics in olives in Italy and to infect other hosts in Europe and Asia. In this study, we report a short protocol to create deletion and complemented mutants using fusion PCR and natural transformation. We also determined the distinct function of two pilin paralogs, the main structural component of TFP involved in twitching motility, which allows this bacterium to move inside the xylem vessels against the flow. One of the paralogs is needed for twitching movement, whereas the other does not have an effect on motility but influences the number and position of TFP. Since twitching motility is fundamental for the virulence of this xylem-limited bacterium, this study contributes to the understanding of the regulation of virulence by this pathogen.

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