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

Author(s): Raye L. Alford, PhD

Gene Cloning II

Genes isolated from genomic DNA by restriction digestion can be cloned into vectors for further analysis. Cloning vectors come in a number of varieties, including phage, plasmid, cosmid, and others. This slide shows a map of the bacterial plasmid vector called pUC19. On the right is the multiple cloning site (purple box labeled MCS). This site contains a number of different restriction enzyme digestion sites, allowing DNA cut by a variety of enzymes to be cloned into this vector. Once inside the bacterial cells, the plasmid and its cloned gene can be cultured to create millions of copies of the target DNA. Plasmids are efficient ways to replicate genes in vitro, but they are limited in that the target DNA cloned into the MCS 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 bacterial or eukaryotic cells can accommodate larger pieces of cloned DNA.

On the left side of the plasmid drawing is an ampicillin resistance gene (ampR). This gene allows any bacteria harboring this plasmid to survive treatment with the antibiotic ampicillin. In this way, cells that have taken up the recircularized plasmid, called transfected, survive in cultures containing ampicillin, while those that do not carry the plasmid die. Therefore, only bacteria carrying the plasmid are grown, enriching the culture for the cloned target DNA. Naturally occurring plasmids, not genetically engineered ones, are the basis for much of the antibiotic resistance seen in bacterial diseases today.