BACKGROUND Seamless ligation cloning extract (SLiCE) is a simple and efficient method for DNA assembly that uses cell extracts from the Escherichia coli PPY strain, which expresses the components of the λ prophage Red/ET recombination system. This method facilitates restriction endonuclease cleavage site-free DNA cloning by performing recombination between short stretches of homologous DNA (≥ 1...

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...

BACKGROUND Recent development of DNA assembly technologies has spurred myriad advances in synthetic biology, but new tools are always required for complicated scenarios. Here, we have developed an alternative DNA assembly method named AFEAP cloning (Assembly of Fragment Ends After PCR), which allows scarless, modular, and reliable construction of biological pathways and circuits from basic gene...

A robust method for the in vivo cloning of large gene clusters was developed based on homologous recombination (HR), requiring only the transformation of PCR products into Escherichia coli cells harboring a receiver plasmid. Positive clones were selected by an acquired antibiotic resistance, which was activated by the recruitment of a short ribosome-binding site plus start codon sequence from t...

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 (MP)-mediated cloning strategy for chimeragenesis and long DNA fragment insertion is presented. MP cloning is a seamless, restriction/ligation-independent method that requires only three steps: i) the fir...

BACKGROUND Restriction-free (RF) cloning, a PCR-based method for the creation of custom DNA plasmids, allows for the insertion of any sequence into any plasmid vector at any desired position, independent of restriction sites and/or ligation. Here, we describe a simple and fast method for performing gene reconstitution by modified RF cloning. RESULTS Double-stranded inserts and acceptors were ...

The need for designing new peptides of biological, pharmacological or clinical importance is increasing. To fulfill this need, a detailed knowledge of the structure-function relationship of known bioactive proteins is a prerequisite, and to obtain such information, we face challenges of inserting and deleting specific sequences into or from known proteins and expressing these modified proteins ...