Multi-fragment site-directed mutagenic overlap extension polymerase chain reaction as a competitive alternative to the enzymatic assembly method

Abstract

Methods for introducing multiple site-directed mutations are important experimental tools in molecular biology. Research areas that use these methods include the investigation of various protein modifications in cellular processes, modifying proteins for efficient recombinant expression, and the stabilization of mRNAs to allow for increased protein expression. Introducing multiple site-directed mutations is also an important tool in the field of synthetic biology. There are two main methods used in the assembling of fragments generated by mutagenic primers: enzymatic assembly and overlap extension polymerase chain reaction (OE-PCR). In this article, we present an improved OE-PCR method that can be used for the generation of large DNA fragments (up to 7.4 kb) where at least 13 changes can be introduced using a genomic template. The improved method is faster (due to fewer reaction steps) and more accurate (due to fewer PCR cycles), meaning that it can effectively compete with the enzymatic assembly method. Data presented here show that the site-directed mutations can be introduced anywhere between 50 and 1800 bp from each other. The method is highly reliable and predicted to be applicable to most DNA engineering when the introduction of multiple changes in a DNA sequence is required.

Stress sensitivity of a fission yeast strain lacking histidine kinases is rescued by the ectopic expression of Chk1 from Candida albicans

“…In addition, to investigate a possible functional conservation between the S. pombe HKs and HKs from an evolutionary divergent yeast species, the HKs from C. albicans were included in the analysis. To allow for a correct protein translation of genes from C. albicans in S. pombe, all the CTG codons (encoding serine in C. albicans and leucine in S. pombe ) had to be changed to TCT (encoding serine in S. pombe ) (Waneskog and Bjerling 2014) (“Materials and methods”). The six genes were cloned into the S. pombe expression vector pREP3X.…”

“…To remove the intron sequence, both flanking exons were PCR amplified with primers [MAK1_F/MAK1_74-173_deletion_R and MAK1_74-173_deletion_F/MAK1_R (Table S2)] creating two exon DNA fragments with 25 bp overlapping sequences. The two DNA fragments were joined together with overlap-extension PCR using a previously described PCR protocol (Waneskog and Bjerling 2014). The resulting mak1 + gene without introns was subsequently cloned into the pCR2.1 TOPO vector (Life Technologies) and sequenced.…”

“…The amplification primers for the mak genes contained a Sal I site to facilitate subcloning and all three genes were cloned into the pREP3X plasmid using Sal I restriction enzyme. The adaption to the universal genetic code and subsequent assembly of CaCHK1 and CaSLN1 using multi-fragment site-directed mutagenic overlap-extension PCR are described in (Waneskog and Bjerling 2014). The CaNIK1 gene was also codon adapted for ectopic expression in fission yeast by changing the two CTG codons (S463 and S899) to TCT codons and performing subsequent assembly using the same protocol as for the CaCHK1 and CaSLN1 assemblies with primer pairs NIK1_F/NIK1_L463S_R, NIK1_L463S_F/NIK1_L899S_R, and NIK1_L899S_F/NIK1_R (Table S2).…”

Cellular Localization and Regulation of Expression of the PLET1 Gene in Porcine Placenta

“…The total RNA isolated from the porcine placentas was used to synthesize the cDNA, and the cDNA was used to amplify the PLET1-S and PLET1-L 3′ UTRs, respectively. The first polyadenylation site-directed mutant was generated by overlap extension PCR method [45]. Primers used were listed in Table S1.…”

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

“…Based on the principle and strategy of OE-PCR in Single-SDM, OE-PCR has been developed for use in Multi-SDM (Urban et al, 1997;Tian et al, 2010;Luo et al, 2012;Wäneskog and Bjerling, 2014). In traditional OE-PCR, two or more mutationembedding fragments are separately amplified using internal primers which contain mutation sites and an overlapping sequence.…”