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Gene cloning means that the extracted or synthesized target genes are spliced and recombined with an autonomously replicating vector gene, and then imported into a recipient cell to fulfill asexual propagation, so that new gene molecules are produced. Since the 1970s when the first recombinant DNA molecular clone was created, gene cloning has become the core technology of molecular biology. This promotes the advancement of molecular biology and rigorously facilitates rapid progress in bio-medicine research and industry.
Firstly, gene cloning is beneficial for precise analysis on expression and regulation of target genes and protein interactions, including component regulation, transcription factor research and non-coding RNA research. Secondly, it can be used to realize mass production of natural proteins that cannot be easily obtained. Thirdly, it can be utilized to shape and alter biological genome structures, making their application prospect more promising. With progress in gene sequencing and analysis demands as well as the next-generation sequencing technique (NGS) in recent years, gene cloning plays an increasingly important role.
Gene cloning can be divided into the following three major categories：
1. Classical gene cloning: Restriction enzyme and ligase are required. As the most extensively applied gene cloning approach, it is user-friendly. Due to limitations of restriction enzyme cutting sites and ligase efficacy, it is only suitable for small-segment gene cloning.
2. Gene cloning requiring modification enzyme: Neither restriction enzyme nor ligase is required. For this reason, flexibility and application space of gene cloning are improved. For example, the Uracil-DNA Glycosylase (UDG) cloning method is featured with simple operation and high cloning efficiency.
3. Gene cloning requiring recombinase: Rapid and efficient targeting cloning is achieved by virtue of recombinase and its specific recognition sites. Based on this method, not only is high-throughput cloning fulfilled, but it is also appropriate for expression vectors in multiple systems such as prokaryote, yeast, insect cells and mammalian cells. At present, the recombinase based gene cloning (e.g., GateWay and Golden Gate) attracts extensive attention.
The classical gene cloning method primarily consists of the following procedures: target gene preparation; digestion and cleavage of target genes and vectors by restriction enzyme; litigation of enzyme cleavage post genes and vectors by DNA ligase; preparation of DNA recombinant; importing he prepared DNA recombinant into a host cell; clone selecting and determination; and, amplification and expressions of genes in host cells, etc.
A mature and complete gene cloning R & D platform has been established in our company. In addition to varieties of cloning vectors and expression vectors, the platform also covers different types of host cells and has a complete set of gene manipulation procedures and standards. In this way, accurate and efficient cloning of the target gene can be ensured. Especially, we are also well experienced in gene codon optimization, expression vector design and construction, and selection of fusion objects and labels. In the end, high-throughput, high-purity and high-stability expressions of various proteins in the prokaryotic expression system can be completed.
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