Article

Rapid gene splicing and multi-sited mutagenesis by one-step overlap extension polymerase chain reaction.

Lauren Biotechnology, Nanjing 210019, China.
Analytical Biochemistry (impact factor: 3). 07/2012; 429(1):76-8. DOI:10.1016/j.ab.2012.06.027 pp.76-8
Source: PubMed

ABSTRACT Gene splicing and site-directed mutagenesis (SDM) are important to introduce desired sequences in target DNA. However, introducing mutations at multiple sites requires multiple steps of DNA manipulation, which is time-consuming and labor-intensive. Here, we present a rapid efficient gene splicing and multi-sited mutagenesis method that introduces mutations at two distant sites via sequential connection of DNA fragments by one-step overlap extension polymerase chain reaction (OE-PCR). This bottom-up approach for DNA engineering can be broadly used to study protein structure-function, to optimize codon use for protein expression, and to assemble genes of interest.

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Keywords

bottom-up approach
 
distant sites
 
DNA fragments
 
Gene splicing
 
genes
 
introduces mutations
 
multi-sited mutagenesis method
 
multiple sites
 
multiple steps
 
one-step overlap extension polymerase chain reaction
 
rapid efficient gene splicing
 
site-directed mutagenesis
 
study protein structure-function
 
time-consuming