Efficient multi-site-directed mutagenesis directly from genomic template
- PDF / 249,492 Bytes
- 5 Pages / 595.276 x 793.701 pts Page_size
- 113 Downloads / 179 Views
1.
Introduction
As a fundamental technique, site-directed mutagenesis is a powerful tool for investigating the function of regulatory DNA sequences such as promoter, intron, 5′- or 3′-untranslated region (UTR) (Higuchi et al. 1988; Ito and Lai 1997). Generally, the desired change is in one site only, but sometimes one needs to simultaneously introduce multiple mutations in different positions in a gene to understand the DNA function or to optimize the gene expression. Among a variety of multi-site-directed mutagenesis (MSM) approaches, overlap extension PCR (OE-PCR) and Quick-change multisite-directed mutagenesis systems developed by Stratagene Company are predominately used owing to their simplicity and efficiency. Quick-change method is simple for MSM, but it requires circular plasmid as an amplification template (Wang and Malcolm 1999; Hogrefe et al. 2002). To use this method, non-mutated target sequence coming from genomic DNA or cDNA must be first cloned into the plasmid, and then the plasmid employed for MSM. Thus, Quick-change method is simple only for plasmid-based mutagenesis. OE-PCR is traditional approaches widely used for sitedirect mutagenesis (Ling and Robinson 1997). Compared to Quick-change method, OE-PCR is advantageous for MSM with respect to linear template. In this method, first, two or Keywords.
more mutation-containing fragments are amplified separately by using one universal and one mutagenic primer or two mutagenic primer pairs. The two or more intermediate products with complementary ends form a new template DNA by allowing the 3′ overlap of each strand to serve as primer for the 3′ extension of the complementary strand. Then the mutant DNA is generated from the fused new template DNA through PCR amplification using two universal primers (Ling and Robinson 1997). In the traditional OE-PCR approach, it is necessary to remove the wild-type DNA template and remaining primers in the first amplification, and so purifying the products of the first PCR reaction by gel electrophoresis is needed prior to overlap extension (Peng et al. 2006). This purification step is laborious and cost-consumptive. Although improved megaprimer-PCR method and asymmetric overlap extension PCR have been reported to bypass the intermediate product purification step, these methods are preferable for single-site mutagenesis (Tyagi et al. 2004; Xiao et al. 2007). Furthermore, most of the reported MEM strategies based on OE-PCR employed plasmid as the test template, and under the condition of genomic template, we confronted many difficulties when using these methods. Thus, it is still necessary to improve the OE-PCR method for MEM and simultaneously to simplify its procedure suitable for genomic template.
Genomic template; multi-site-directed mutagenesis; overlap extension; PCR; 3′-UTR
http://www.ias.ac.in/jbiosci
Published online: 29 September 2012
J. Biosci. 37(6), December 2012, 965–969, * Indian Academy of Sciences
965
966
Fengtao Luo et al.
In this study, we present an efficient OE-PCR method for MSM directly
Data Loading...
