Francisella tularensis is one of the most deadly bacterial agents, yet most of the genetic determinants of pathogenesis are still unknown. We have developed an efficient targeted mutagenesis strategy in the model organism F. tularensis subsp. novicida by utilizing universal priming of optimized antibiotic resistance cassettes and splicing by overlap extension (SOE). This process enables fast an...

In the last several years, the use of double-stranded DNA templates together with thermostable-polymerase PCR has essentially replaced the use single-stranded DNA templates using the thermolabile polymerase for in vitro mutagenesis. Numerous PCR methods are now available, such as overlap-extension PCR, megaprimer PCR, and inverse PCR. All these PCR methods are reliable, effective, and convenien...

This article describes a one-step procedure based on Taq polymerase for the precise assembly of DNA fragments into circular constructs as long as 6 kb. The only prior step needed was the amplification of the gene to be cloned and the linear vector backbone, and the whole process up to assembly and circularization lasted only 2 days, compared with the conventional method's 2 weeks. Furthermore, ...

Splicing by directed ligation (SDL) is a method of in-phase joining of PCR-generated DNA fragments that is based on a pre-designed combination of class IIS restriction endonuclease recognition and cleavage sites. Since these enzymes cleave outside of their recognition sites, the resulting sticky end can have any desired sequence, and the site itself can be removed and does not appear in the fin...

The Pichia pastoris expression system was used to produce recombinant human erythropoietin, a protein synthesized by the adult kidney and responsible for the regulation of red blood cell production. The entire recombinant human erythropoietin (rhEPO) gene was constructed using the Splicing by Overlap Extension by PCR (SOE-PCR) technique, cloned and expressed through the secretory pathway of the...

The synthesis of a gene coding for horseradish peroxidase (HRP, isozyme c; EC 1.11.1.7) is described using a polymerase chain reaction (PCR)-mediated gene synthesis approach developed in our laboratory. In this approach, all the oligonucleotides making up the gene are ligated in a single step by using the two outer oligonucleotides as PCR primers and the crude ligation mixture as the target. Th...

Four exons of the human genomic GM-CSF gene were assembled together using gene splicing by overlap extension (SOE) method. The resulting nucleotide sequence was cloned in the pET23a(+) ex‌pression vector under the control of strong bacteriophage T7 transcription and translation signals. The construct obtained was Transferred into the E. coli strain, BL21(DE3) pLysS and IPTG was used for inducti...