title: Thermostable DNA ligase-mediated PCR production of circular plasmid (PPCP) and its application in directed evolution via in situ error-prone PCR.
abstract: Polymerase chain reaction (PCR) is a powerful method to produce linear DNA fragments. Here we describe the Tma thermostable DNA ligase-mediated PCR production of circular plasmid (PPCP) and its application in directed evolution via in situ error-prone PCR. In this thermostable DNA ligase-mediated whole-plasmid amplification method, the resultant DNA nick between the 5' end of the PCR primer and the extended newly synthesized DNA 3' end of each PCR cycle is ligated by Tma DNA ligase, resulting in circular plasmid DNA product that can be directly transformed. The template plasmid DNA is eliminated by 'selection marker swapping' upon transformation. When performed under an error-prone condition with Taq DNA polymerase, PPCP allows one-step construction of mutagenesis libraries based on in situ error-prone PCR so that random mutations are introduced into the target gene without altering the expression vector plasmid. A significant difference between PPCP and previously published methods is that PPCP allows exponential amplification of circular DNA. We used this method to create random mutagenesis libraries of a xylanase gene and two cellulase genes. Screening of these libraries resulted in mutant proteins with desired properties, demonstrating the usefulness of in situ error-prone PPCP for creating random mutagenesis libraries for directed evolution.
authors: Biofuels Institute, School of Environment, Jiangsu University, Zhenjiang, Jiangsu 212013, PR China.
authors: Yilin
authors: Y
authors: Le
authors: Huayou
authors: H
authors: Chen
authors: Robert
authors: R
authors: Zagursky
authors: J H David
authors: JH
authors: Wu
authors: Weilan
authors: W
authors: Shao
mesh_terms: D001203:Ascomycota / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D044602:Cellulases / Q000235:genetics / Q000378:metabolism; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D019020:Directed Molecular Evolution / Q000379:methods*; D043364:Endo-1,4-beta Xylanases / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D006358:Hot Temperature; D016296:Mutagenesis; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D019914:Taq Polymerase / Q000378:metabolism; D020124:Thermotoga maritima / Q000737:chemistry* / Q000201:enzymology / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D005656:Fungal Proteins; D019914:Taq Polymerase; D044602:Cellulases; D043364:Endo-1,4-beta Xylanases; D011088:DNA Ligases; D000072481:DNA Ligase ATP
keywords: amplification of circular plasmids; directed evolution; error-prone PCR; random mutagenesis libraries; thermostable DNA ligase
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