OverFlap PCR – a reliable approach for generation of plasmid DNA libraries containing random sequences without template bias.

Over the decades the improvement of naturally occurring proteins and creation of novel ones has been the primary goal for many practical biotechnology researchers and it is widely recognized that randomization of protein sequences coupled to various effect screening methodologies is one of the most powerful techniques for fast, efficient and purposeful approach for acquisition of desired improvements. Over the years considerable advancements have been made in this field, however development of PCR based or template guided methodologies has been hampered by the resulting template sequence bias. In this article we present novel whole plasmid amplification based approach, which we named OverFlap PCR, for randomization of virtually any region of the plasmid DNA, without introduction of mentioned bias.

Developing Qualitative Plasmid DNA Reference Materials to Detect Mechanisms of Quinolone and Fluoroquinolone Resistance in Foodborne Pathogens

The aim of this study was to develop homogeneous and stable plasmid DNA reference materials for detecting the mechanisms of resistance to quinolones and fluoroquinolones in foodborne pathogens. The DNA fragments of 11 target genes associated with quinolone and fluoroquinolone resistance were artificially synthesized, inserted into plasmid vectors, and transferred into recipient cells. PCR and sequencing of DNA were performed to assess the genetic stability of the target DNA in recombinant Escherichia coli DH5α cells during subculturing for 15 generations. The limit of detection (LOD) of the target DNA was determined using PCR and real-time qualitative PCR (qPCR). The homogeneity and storage stability of plasmid DNA reference materials were evaluated in terms of plasmid DNA quantity, PCR-measured gene expression, and qPCR threshold cycle. All 11 target DNAs were successfully synthesized and inserted into vectors to obtain recombinant plasmids. No nucleotide mutations were identified in the target DNA being stably inherited and detectable in the corresponding plasmids during subculturing of recombinant strains. When the target DNA was assessed using PCR and qPCR, the LOD was ≤1.77 × 105 and 3.26 × 104 copies/μL, respectively. Further, when the reference materials were stored at 37 °C for 13 days, 4 °C for 90 days, and −20 °C for 300 days, each target DNA was detectable by PCR, and no mutations were found. Although the threshold cycle values of qPCR varied with storage time, they were above the LOD, and no significant differences were found in the quantity of each plasmid DNA at different timepoints. Further, the homogeneity and stability of the materials were highly consistent with the requirements of standard reference materials. To summarize, considering that our plasmid DNA reference materials conformed to standard requirements, they can be used to detect the mechanisms of quinolone and fluoroquinolone resistance in foodborne pathogens.

Comparative Plasmid-Mediated Molecular Resistance Status of Diarrheic Escherichia coli Isolates from Human and Goat Kids

Globally antibiotic resistance has become a major concern, which warrants the real time monitoring for resistance in very common pathogenic organisms. E. coli is normal micro flora in humans, but sometimes it can be pathogenic. For the observation and increment of antimicrobial resistance among pathogen, E. coli has been one of the important pathogens. It is present everywhere in fecal, water, food etc., if resistant E. coli will present in the environment that it can be transferrable anywhere through water, fecal food, animals and humans. This is very dangerous to living beings. This study was designed on status of antibiotic resistance in E. coli isolates in human kids and animal kids, both. Newborns are affected more because of poor or lack of immune system. In this study, fecal materials were used as sample material collected from goat kids (0-3 months) and human children (up to 3 years) residing in same local area. Fifteen fecal samples were collected from human children (up to 3 years) and goat kids (0-3 months) in each case to study the risk of transmission of resistance in E. coli isolates. PCR was conducted on genomic DNA isolates for the presence of usp A gene of E. coli. Multiplex PCR were conducted on plasmid DNA isolates for the resistance specific genes. Molecular resistance results in goat kids isolates showed resistance to antibiotics with tetracycline, sulphonamide, gentamycin, streptomycin and cephalothin to the level of 93.33, 53.33, 46.66, 13.33 & 6.66% respectively, whereas, human E. coli isolates were showed the highest resistance to sulphonamide, Tetracycline and β-lactams were as 53.33, 46.66 and 13.33% respectively but no resistance with gentamycin and streptomycin. Here, we concluded that humans and animals both were refractory to the various groups of antibiotics. This study will help in making the strategy for prevention or reduction of resistance in public

Fabrication and Biological Activities of Plasmid DNA Gene Carrier Nanoparticles Based on Biodegradable l-Tyrosine Polyurethane

Gene therapy is a suitable alternative to chemotherapy due to the complications of drug resistance and toxicity of drugs, and is also known to reduce the occurrence of cellular mutation through the use of gene carriers. In this study, gene carrier nanoparticles with minimal toxicity and high transfection efficiency were fabricated from a biocompatible and biodegradable polymer, l-tyrosine polyurethane (LTU), which was polymerized from presynthesized desaminotyrosyl tyrosine hexyl ester (DTH) and polyethylene glycol (PEG), by using double emulsion and solvent evaporation techniques, resulting in the formation of porous nanoparticles, and then used to evaluate their potential biological activities through molecular controlled release and transfection studies. To assess cellular uptake and transfection efficiency, two model drugs, fluorescently labeled bovine serum albumin (FITC-BSA) and plasmid DNA-linear polyethylenimine (LPEI) complex, were successfully encapsulated in nanoparticles, and their transfection properties and cytotoxicities were evaluated in LX2 as a normal cell and in HepG2 and MCF7 as cancer cells. The morphology and average diameter of the LTU nanoparticles were confirmed using light microscopy, transmission electron microscopy, and dynamic light scattering, while confocal microscopy was used to validate the cellular uptake of FITC-BSA-encapsulated LTU nanoparticles. Moreover, the successful cellular uptake of LTU nanoparticles encapsulated with pDNA-LPEI and the high transfection efficiency, confirmed by gel electrophoresis and X-gal assay transfection, indicated that LTU nanoparticles had excellent cell adsorption ability, facilitated gene encapsulation, and showed the sustained release tendency of genes through transfection experiments, with an optimal concentration ratio of pDNA and LPEI of 1:10. All the above characteristics are ideal for gene carriers designed to transport and release drugs into the cytoplasm, thus facilitating effective gene therapy.