Bioinformatic Analysis in Designing Mega-primer in Overlap Extension PCR Cloning (OEPC) Technique

Bioinformatics has developed into an application tool for basic and applied research in the biomedical and biotechnology field. Polymerase Chain Reaction (PCR) is a common technique in the molecular area that has always involved bioinformatics science. PCR cloning techniques such as TA cloning and PCR-mediated cloning exhibit complex processes with low success rates. One easy, effective, and practical solution is to use a mega-primer with the Overlap Extension PCR Cloning (OEPC) technique. The success of PCR cloning using the mega-primer design in the OEPC technique is strongly influenced by the characteristics of the mega-primer used. Knowledge of mega-primer characteristics is one of the important factors in the success of PCR cloning. The design process for the mega-primer str promoter was characterized based on the principle of a genetic algorithm using the web-based bioinformatics tools such as ClustalW, NetPrimer, and BLAST. The success of the mega-primer construction in producing recombinant pSB1C3 vector has been confirmed by the sequencing method and the function of the reporting protein (AmilCP). DNA analysis shows a 100 % homologous sequence on the str promoter, while E. coli colonies successfully express the purplish-blue color. Mega-primer characters can save costs and time of the research by maintaining the primer parameters that provide optimal values and increase the success value of PCR cloning via bioinformatics software. Hence, implications on biological problems, especially using DNA and amino acid sequences, could solve rapidly.

An improved gene synthesis method with asymmetric directions of oligonucleotides designed using a simulation program

Artificial gene synthesis based on oligonucleotide augmentation is known as overlap extension PCR which generates a variety of intermediate synthetic products. The orientation and concentration of oligomers can be adjusted to reduce the synthesis of intermediates and optimize the full-length process of DNA synthesis, using a simulation program for serial oligomer extension. The efficiency of the serial oligomer extension process is predicted to be greatest when oligomers are in a ‘forward-reverse-reverse-reverse’ direction. Oligomers with such designed directions demonstrated generation of the desired product in the shortest time (number of cycles) by repeated annealing and elongation. This method, named Asymmetric Extension supported by a Simulator for Oligonucleotide Extension (AESOE), has shown efficiency and effectiveness with potentials for future improvements and optimal usage in DNA synthesis.

An improved overlap extension PCR for simultaneous multiple sites large fragments insertion, deletion and substitution

The existing molecular cloning methods are often limited by the availability of suitable restriction sites. It is still a challenge for simultaneous cloning of multiple fragments into different sites of a single vector. Here we developed a novel method named improved overlap extension PCR (IOEP) for restriction enzyme independent cloning of large fragments. The addition of primers enables IOEP to exponentially amplify the overlap extension product, thus greatly improves the amplification efficiency of large fragments. Moreover, coupled with the benefit of T4 DNA polymerase to improve cloning efficiency, our method can be used to simultaneously insert, delete and replace multiple DNA fragments at different sites.

A Short Protocol for Gene Knockout and Complementation in Xylella fastidiosa Shows that One of the Type IV Pilin Paralogs (PD1926) Is Needed for Twitching while Another (PD1924) Affects Pilus Number and Location

ABSTRACT Twitching motility is one of the major virulence factors of the plant-pathogenic bacterium Xylella fastidiosa, and it is mediated by type IV pili (TFP) that are present at one of the cell poles. Genome analysis of X. fastidiosa showed the presence of at least four paralogs of the gene pilA, which encodes the TFP major pilin subunit. However, whether all of these paralogs have a functional role in TFP structure and function is unknown. Here, using a short and reliable protocol based on overlap extension PCR and natural transformation, deletion mutants of two pilA paralogs (pilA1 PD1924 and pilA2 PD1926) were generated in two X. fastidiosa subsp. fastidiosa strains, WM1-1 and TemeculaL, followed by assessment of twitching motility and biofilm formation. Deletion of pilA2 caused loss of twitching motility, whereas deletion of pilA1 did not influence twitching motility but caused hyperpiliation and extended distribution of TFP along the sides of the cell. Loss of twitching motility due to pilA2 deletion was restored when a wild-type copy of the pilA2 gene was added at a neutral site in the genome of mutants in both wild-type backgrounds. This study demonstrates that PCR templates generated by overlap extension PCR can be successfully used to rapidly generate gene knockouts and perform genetic complementation in X. fastidiosa, and that twitching motility in X. fastidiosa is controlled by regulating the transcription of the major pilin subunit, pilA2. IMPORTANCE The bacterial plant pathogen Xylella fastidiosa causes incurable diseases in multiple hosts, including grape, citrus, and blueberry. Historically restricted to the Americas, it was recently found to cause epidemics in olives in Italy and to infect other hosts in Europe and Asia. In this study, we report a short protocol to create deletion and complemented mutants using fusion PCR and natural transformation. We also determined the distinct function of two pilin paralogs, the main structural component of TFP involved in twitching motility, which allows this bacterium to move inside the xylem vessels against the flow. One of the paralogs is needed for twitching movement, whereas the other does not have an effect on motility but influences the number and position of TFP. Since twitching motility is fundamental for the virulence of this xylem-limited bacterium, this study contributes to the understanding of the regulation of virulence by this pathogen.

Site-directed mutagenesis in Brucella abortus S19 by overlap extension PCR-based procedure

Introduction of a site-directed mutation can be effective method to evaluate properties of various genes. Brucellosis is one of the most common zoonotic infectious diseases, which causes great economic losses. Thus, determination of pathogenicity factors in the genus Brucella can lead to the control of this health problem. Due to the importance of site-directed mutations in identification of genomic structure, overlap extension polymerase chain reaction (PCR) has been introduced as an improved technique for the removal and replacement of gene targets. In this study, three DNA fragments were amplified and combined using a two-step PCR with specific primers. The resulting fusion PCR product, obtained without any change in the nucleotide sequence, was cloned in a specific position in the pBluescript II SK (-) plasmid using restriction enzymes. Finally, the construct was transferred into cells of Brucella abortus S19 by electroporation and replaced the target gene (wbkA) in the genome of the bacterium. PCR analysis was performed on kanamycin-resistant colonies to provide genetic evidence that the B. abortus wbkA gene was interrupted by the kanamycin cassette. The results of this study show that the optimized modified technique, splicing by overlap extension PCR, is effective in creating mutations in the bacterial genome and can easily be used in Brucella spp.