DNA cloning is an essential technique in life sciences research, which is used to make multiple, identical copies of a particular piece of DNA. Most of these DNA fragments are produced by digestion of existing DNA fragments by restriction endonuclease enzymes, or by PCR amplification. Cloning involves preparation of vectors and inserted DNA, ligation of the insert into the vector, transformation of cell of interest, and identification of positive clones.
High quantities of DNA of interest exist in animal tissues, cells, bacterial cultures, etc., and are generally obtained through conventional extraction techniques. After successful isolation, the DNA of interest is ligated to a plasmid vector. A plasmid is a small, circular, double-stranded DNA molecule that replicates independently of the chromosomal DNA in bacteria. Plasmids used in the laboratory represents a smaller version derived from larger plasmids that occur naturally in bacteria, which contain the origin of replication, a drug-resistant gene, and unique restriction sites that facilitate the insertion of DNA fragments. The selection of restriction enzymes is critical in the design of cloning strategies. Restriction enzymes recognize the specific sequences in the DNA and cleave at their recognition sites to generate double-stranded breaks. Fragments with either a 5' or 3' overhang are referred to as sticky ends, while those without overhang are referred to as blunt ends. These complementary sticky ends are easily paired, increasing the efficiency of the ligation reaction and thus the chances of successful cloning. A complete circular plasmid DNA insert is obtained by ligating either sticky or blunt ends of DNA insert into a plasmid using DNA ligases (e.g., T4 DNA Ligase). The recombinant plasmid is introduced into a bacterial host for replication. The inserted DNA fragment is also copied along with the rest of the bacterial DNA.
DNA cloning is the starting point for many genetic engineering approaches to fundamental biological research. Its value lies in the preparation of DNA fragments containing a specific gene, thereby advancing knowledge of the structure, function, expression, and activity control of that gene. A great amount of DNA through DNA cloning makes it possible to produce proteins in large quantities, analyze genetic material in detail or develop gene therapy. Amerigo Scientific offers superior performance and ease-to-use products for molecular cloning. Our DNA cloning kits provide an efficient, powerful, and easy way to obtain the best results. Different vectors are available for Blunt-end, TA or universal DNA cloning to meet different research needs. Additionally, we offer chemically competent cells for routine subcloning procedures, as well as many compounds needed in DNA cloning.
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