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Molecular Basis of Heredity: Part 4. Gene Identification and Tests

Author(s): Raye L. Alford, PhD

Gene Cloning I

Genes isolated from genomic DNA by restriction digestion can be cloned into vectors (bacteriophage, plasmid, cosmid) for further evaluation in the laboratory. Vectors are small, independently replicating DNA molecules carried by viruses or bacteria that can be manipulated in the laboratory to  carry, and copy, genes of interest to researchers. In the cloning process, target DNA is cut by a restriction enzyme. The vector DNA is cut by the same enzyme. The target and vector DNA are mixed together and DNA ligase is added, to join the ends of the target DNA to the ends of the vector DNA. After ligation, the vector is once again a circle but now it carries a piece of the target DNA inserted into the cloning site. The recombinant vector is then transferred into cells in a process called transfection. Once inside the cells, the vector and its cloned gene can be cultured to create millions of copies of the target DNA. Plasmids that replicate in bacterial cells are an efficient mechanism by which genes can be replicated in vitro, but they are limited in that the target DNA cloned into the cloning site can only be of a certain size. Fragments too large are not able to be propagated by the bacterial cells efficiently. Other vectors such as cosmids and phages that replicate in either bacteria or in eukaryotic cells can accommodate larger pieces of cloned DNA.