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...

To clone genes of interest into suitable vectors is the first step to investigate their functions in vitro and in vivo. At the genome era, the sequences of more and more genes were decoded and available gradually. Therefore, it is critical to develop high efficient strategies for cloning genes of interest into different vectors to facilitate the functional analyses of them. In our previous stud...

Heterologous expression has been proven to be a valid strategy for elucidating the natural products produced by gene clusters uncovered by genome sequencing projects. Efforts have been made to efficiently clone gene clusters directly from genomic DNA and several approaches have been developed. Here, we present an alternative strategy based on the site-specific recombinase system Cre/loxP for di...

We describe a novel cloning method termed SLiCE (Seamless Ligation Cloning Extract) that utilizes easy to generate bacterial cell extracts to assemble multiple DNA fragments into recombinant DNA molecules in a single in vitro recombination reaction. SLiCE overcomes the sequence limitations of traditional cloning methods, facilitates seamless cloning by recombining short end homologies (≥15 bp) ...

introduction: beta thalassemias are a heterogenous group of autosomal recessive disorders, characterized by reduced or absent production of the b-globin chain by the affected allele. transplantation of allogenic hematopoietic stem cells (hsc) is a curative approach but this therapeutic option is not available to the majority of patients. transplantation of genetically corrected autologous hsc i...

As a way to circumvent the quantum no-cloning theorem, approximate quantum cloning protocols have received wide attention with remarkable applications. Copying of quantum states to memory qubits provides an important strategy for eavesdropping in quantum cryptography. We report an experiment that realizes cloning of quantum states from an electron spin to a nuclear spin in a hybrid solid-state ...

A novel cloning strategy, replicon rescue, was developed for cloning genes disrupted by plasmid insertions. After ligation to a tetracycline resistance cassette, fragments containing a bacterial origin of replication from the insertion are recovered in Escherichia coli because they replicate autonomously. Restriction enzymes for cloning are so chosen that the only legitimate two fragment ligati...

Fusion protein technology represents the strategy to achieve rapid, efficient, and cost-effective proteinexpression. Epsilon and Beta toxins are the most potent Clostridial toxins and cause disease in animals.This study describes in silico fusion of Clostridium perfringens types D and B epsilon and beta toxin genesthat was used for cloning in E.coli. The etx and cpb genes were...

Cloning is an essential prerequisite to test protein design and engineering ideas. However, it is often time consuming, unreliable, and therefore frustrating. Here, we present a streamlined cloning strategy that incorporates a powerful white and green screening protocol to identify colonies with inserts. We use circular polymerase extension cloning, which is both ligation and sequence independe...

Forward genetic screens have been used as a powerful strategy to dissect complex biological pathways in many model systems. A significant limitation of this approach has been the time-consuming and costly process of positional cloning and molecular characterization of the mutations isolated in these screens. Here, the authors describe a strategy using microarray hybridizations to facilitate pos...