Molecular cloning methods based on primer and overlap-extension PCR are widely used due to their simplicity, reliability, low cost and high efficiency. In this article, an efficient mega primer-mediated (MP) cloning strategy for chimaeragenesis and long DNA fragment insertion is presented. MP cloning is a seamless, restriction/ligation-independent method that requires only three steps: (i) the ...

background: hepatitis c virus (hcv) infection is one of the most prevalent infectious diseases responsible for high morbidity and mortality worldwide. therefore, designing new and effective therapeutics is of great importance. the aim of the current study was to construct a dna vaccine containing structural proteins of hcv and evaluation of its expression in a eukaryotic system. methods: struct...

Bacterial artificial chromosome (BAC) and P1-derived artificial chromosome (PAC) systems were previously developed for cloning of very large eukaryotic DNA fragments in bacteria. We report the feasibility of cloning very large fragments of eukaryotic DNA in bacteria using conventional plasmid-based vectors. One conventional plasmid vector (pGEM11), one conventional binary plasmid vector (pSLJ17...

Synthetic biology and genome-scale protein work both require rapid and efficient cloning, expression and purification. Tools for co-expression of multiple proteins and production of fusion proteins with purification and solubility tags are often desirable. Here we present a survey of plasmid vectors that provide for some of these features with a focus on tools for rapid cloning and traceless ta...

The precise assembly of specific DNA sequences is a critical technique in molecular biology. Traditional cloning techniques use restriction enzymes and ligation of DNA in vitro, which can be hampered by a lack of appropriate restriction-sites and inefficient enzymatic steps. A number of ligation-independent cloning techniques have been developed, including polymerase incomplete primer extension...

Aeromonas hydrophila is a gram-negative bacterium which associated with gastrointestinal diseases and septicaemia. This pathogenic bacterium has several virulence factors ranging from pili to the excreted protein which called (Aerolysin) with minor and major effects, respectively. Additionally, Aeromonas hydrophila is a widely distributed bacterium that commonly causes ulcers in cyprinid fish s...

Background and Aims: NDV (Newcastle Disease Virus) is one of the viruses that cause disease in avian with severe economic losses in the poultry industry in many countries. Fusion protein (F) which plays a major role in the virus pathogenicity contains several regions that have a role in the fusion process. Mutation in the sequence of HR1 & HR2 regions of this protein prevents fusion of the viru...

Mycophenolic acid (MPA) is a fungal metabolite possessing antiviral, antifungal, antibacterial, antitumor and anti-psoriasis activities. It is being used as an immunosuppressive agent in kidney, heart and liver transplantation patients. In the presence of MPA, the proliferation of the B and T lymphocytes is inhibited. The MPaB and MPaE genes reside in a 25 kb gene cluster in t...

BACKGROUND Molecular cloning of functional immunoglobulin genes from single plasma cells is one of the most promising technologies for the rapid development of monoclonal antibody drugs. However, the proper insertion of PCR-amplified immunoglobulin genes into expression vectors remains an obstacle to the high-throughput production of recombinant monoclonal antibodies. RESULTS We developed a s...

Transformation-associated recombination (TAR) is a cloning technique that allows specific chromosomal regions or genes to be isolated directly from genomic DNA without prior construction of a genomic library. This technique involves homologous recombination during spheroplast transformation between genomic DNA and a TAR vector that has 5' and 3' gene targeting sequences (hooks). Typically, TAR ...