scholarly journals Optimization of overlap extension PCR for efficient transgene construction

MethodsX ◽  
2020 ◽  
Vol 7 ◽  
pp. 100759 ◽  
Author(s):  
Roland S. Hilgarth ◽  
Thomas M. Lanigan
Keyword(s):  
Overlap Extension Pcr ◽  
Overlap Extension

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.

scholarly journals Simple Method for Markerless Gene Deletion in Multidrug-Resistant Acinetobacter baumannii

2015 ◽  
Vol 81 (10) ◽  
pp. 3357-3368 ◽  
Author(s):  
Man Hwan Oh ◽  
Je Chul Lee ◽  
Jungmin Kim ◽  
Chul Hee Choi ◽  
Kyudong Han
Keyword(s):  
Antibiotic Resistance ◽  
Acinetobacter Baumannii ◽  
Gene Deletion ◽  
Multidrug Resistant ◽  
Simple Method ◽  
Content Type ◽  
Overlap Extension Pcr ◽  
Overlap Extension ◽  
Restriction Sites ◽  
Selection Of

ABSTRACTThe traditional markerless gene deletion technique based on overlap extension PCR has been used for generating gene deletions in multidrug-resistantAcinetobacter baumannii. However, the method is time-consuming because it requires restriction digestion of the PCR products in DNA cloning and the construction of new vectors containing a suitable antibiotic resistance cassette for the selection ofA. baumanniimerodiploids. Moreover, the availability of restriction sites and the selection of recombinant bacteria harboring the desired chimeric plasmid are limited, making the construction of a chimeric plasmid more difficult. We describe a rapid and easy cloning method for markerless gene deletion inA. baumannii, which has no limitation in the availability of restriction sites and allows for easy selection of the clones carrying the desired chimeric plasmid. Notably, it is not necessary to construct new vectors in our method. This method utilizes direct cloning of blunt-end DNA fragments, in which upstream and downstream regions of the target gene are fused with an antibiotic resistance cassette via overlap extension PCR and are inserted into a blunt-end suicide vector developed for blunt-end cloning. Importantly, the antibiotic resistance cassette is placed outside the downstream region in order to enable easy selection of the recombinants carrying the desired plasmid, to eliminate the antibiotic resistance cassette via homologous recombination, and to avoid the necessity of constructing new vectors. This strategy was successfully applied to functional analysis of the genes associated with iron acquisition byA. baumanniiATCC 19606 and toompAgene deletion in otherA. baumanniistrains. Consequently, the proposed method is invaluable for markerless gene deletion in multidrug-resistantA. baumannii.

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