A novel megaprimed and ligase-free, PCR-based, site-directed mutagenesis method

The terminal enzymes of cholesterol synthesis, DHCR24 and DHCR7, interact physically and functionally

“…A plasmid encoding the human DHCR24 sequence (NM_014762.3) containing a Flp recombinase target (FRT) recombination site (pcDNA5-DHCR24-V5/FRT) ( 19 ) was used to create the DHCR24 mutant, pcDNA5-DHCR24 Y471S-V5/FRT, using megaprimed site-directed mutagenesis ( 20 ). This was then used to generate the CHO-DHCR24 Y471S stable cell-line.…”

An easy-to-use site-directed mutagenesis method with a designed restriction site for convenient and reliable mutant screening

“…Site-directed mutagenesis (SDM) has a variety of applications and is extensively used in molecular biology. Over the last three decades, various SDM methods have been described (Nagy et al, 2004;Zheng et al, 2004;Seyfang and Jin, 2004;An et al, 2005;Wei et al, 2004;Jin et al, 2007;Heckman and Pease, 2007;Tseng et al, 2008;Li et al, 2008;Chapnik et al, 2008) and some commercial SDM kits based on these techniques are available. The SDM techniques can be grouped into two major categories: polymerase chain reaction (PCR)-based and non-PCR-based.…”

Strategies used for genetically modifying bacterial genome: site-directed mutagenesis, gene inactivation, and gene over-expression

“…With the development of genetic engineering technology, numerous novel Single-SDM methods based on the QuikChange™ SDM system (QCM)-like kits or previous protocols have been developed to overcome the disadvantages of previous methods (Ke and Madison, 1997;Urban et al, 1997;Salerno et al, 2005;Qi and Scholthof, 2008;Tseng et al, 2008;Wang et al, 2011;Adachi and Fukuhara, 2012;Wan et al, 2012;Sun et al, 2015). Most of these methods can be widely used to construct various difficult-toconstruct mutants in areas where traditional SDM protocol cannot perform.…”

“…2.1.3 Single-SDM by TA strategy Although these PCR-based approaches are useful and powerful for SDM, there are still many defects: (1) long or HPLC-purified primer (Davis et al, 1999;Salerno et al, 2005); (2) high-fidelity thermostable DNA polymerase and restriction endonuclease (Salerno et al, 2005); (3) the need for subcloning the desired gene into the original plasmid to form a targeted plasmid (Davis et al, 1999); (4) undesired amplification of the wild-type gene template (Ke and Madison, 1997); (5) the self-annealing of megaprimer strands (Brøns-Poulsen et al, 1998;Siloto and Weselake, 2012); (6) the unbalanced melting temperatures of the primers (Tseng et al, 2008;Saeedi et al, 2012); (7) the requirement for a high concentration of megaprimer to ensure the successful modification (Wan et al, 2012); (8) the requirement for additional steps for preparation of the template plasmid (Urban et al, 1997) or restriction endonuclease treatments of PCR products (Seraphin and KandelsLewis, 1996).…”