Simple Cloning by Prolonged Overlap Extension-PCR with Application to the Preparation of Large-Size Random Gene Mutagenesis Library in Escherichia coli

2016 ◽  
pp. 49-61 ◽  
Author(s):  
Chao Zhong ◽  
Chun You ◽  
Ping Wei ◽  
Yi-Heng Percival Zhang
Keyword(s):  
Escherichia Coli ◽  
Overlap Extension Pcr ◽  
Large Size ◽  
Overlap Extension

scholarly journals Simple Cloning via Direct Transformation of PCR Product (DNA Multimer) to Escherichia coli and Bacillus subtilis

2011 ◽  
Vol 78 (5) ◽  
pp. 1593-1595 ◽  
Author(s):  
Chun You ◽  
Xiao-Zhou Zhang ◽  
Y.-H. Percival Zhang
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Restriction Enzyme ◽  
Dna Fragments ◽  
Direct Transformation ◽  
Content Type ◽  
Overlap Extension Pcr ◽  
Pcr Product ◽  
Overlap Extension ◽  
General Restriction

ABSTRACTWe developed a general restriction enzyme-free and ligase-free method for subcloning up to three DNA fragments into any location of a plasmid. The DNA multimer generated by prolonged overlap extension PCR was directly transformed inEscherichia coli[e.g., TOP10, DH5α, JM109, and BL21(DE3)] andBacillus subtilisfor obtaining chimeric plasmids.

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 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.

Overlap Extension PCR Cloning

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

scholarly journals Expression of Human Salivary Histatin and Cystatin/ Histatin Chimeric cDNAs in Escherichia coli

1993 ◽  
Vol 4 (3) ◽  
pp. 581-590 ◽  
Author(s):  
L.A. Bobek ◽  
H. Tsai ◽  
M.J. Levine
Keyword(s):  
Escherichia Coli ◽  
Fusion Proteins ◽  
Expression System ◽  
Full Length ◽  
Blue Staining ◽  
Coomassie Blue Staining ◽  
Coomassie Blue ◽  
Overlap Extension ◽  
Cystatin Sn ◽  
The Individual

We have previously constructed recombinants encoding the full-length and truncated forms of cystatin-SN and expressed these in the Escherichia coli expression system pGEX-2T, which expresses foreign sequences as fusion proteins with glutathione S-transferase (GST). Recombinant cystatins were produced and purified in large quantities. The full-length recombinant cystatin-SN exhibited comparable biological activity and secondary structure to natural cystatin, validating the use of the full-length and mutant recombinant proteins for structure-function studies of salivary molecules. In this study, we have expressed histatin-1 cDNA in the pGEX-3X vector and cystatin-SN/histatin-1 or cystatin-SN/histatin-3 chimeric cDNAs in the pGEX-2T vector. Gene splicing by overlap extension (SOE), a PCR-based method, was used for generating the chimeric cDNAs. Each construct was analyzed by DNA sequencing, which showed the correct junctions and reading frames between the GST/histatin-1 and the GST/cystatin/histatin cDNAs. Expression of histatin and cystatin/histatin chimeras was induced by IPTG and the production of the fusion proteins monitored by SDS-PAGE/Coomassie blue staining and in the case of the GST/cystatin/histatin fusion proteins, also by Western blot using anti-cystatin antibody. The results of these studies showed that we have successfully constructed recombinants encoding the individual and chimeric salivary molecules and efficiently expressed these in E. coli expression system pGEX. Purification and characterization of recombinant histatin and cystatin-histatin hybrid proteins are presently ongoing.

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