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Gene cloning

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  • 1.Gene Cloning A.Arputha Selvaraj

2. DNA Cloning: An Overview DNA technology has launched a revolution in Biotechnology. DNA technology (via gene manipulation) makes it possible to clone genes for basic research and commercial applications. DNA technology is applied to areas ranging from agriculture to criminal law. 3. DNA Cloning Techniques Techniques for gene cloning enable scientists to prepare multiple copies of DNA pieces. DNA pieces are stored in DNA libraries for easy idenfication and accessibility. DNA cloning can occur (in vitro) via Recombinant DNA Technology and Polymerase Chain Reaction (PCR). 4. Recombinant DNA Technology: A closer look Recombinant DNA technology requires two genes (a human gene and a bacterial gene) combine in vitro into one molecule (a cloning vector). The cloning vector is inserted into a bacterial cell and replicated multiple times. 5. Recombinant DNA Cloning The process of cloning a human gene in a bacterial plasmid can be divided into five steps. Copyright 2002 Pearson Education, Inc., publishing as Benjamin Cummings 6. STEP 1. Isolation of vector and gene-source DNA. The source DNA comes from human tissue cells. The source of the plasmid is typically E. coli. The human DNA and the E. coli plasmid are cut using a restriction enzyme. 7. Restriction Enzymes At Work. Restriction enzymes cleave DNA at specific sites to isolate genes of interest. Copyright 2002 Pearson Education, Inc., publishing as Benjamin Cummings 8. STEP 2. Insertion of DNA into the vector. By digesting the plasmid and human DNA with the same restriction enzyme, both DNA pieces can be combined easily. After mixing, both complementary pairs are joined by DNA ligase. This creates a mixture of recombinant DNA molecules. 9. STEP 3. Introduction of the cloning vector into cells. Bacterial cells take up the recombinant plasmids by transformation. This creates a diverse pool of bacteria, some bacteria that have taken up the desired recombinant plasmid DNA, other bacteria that have taken up other DNA, both recombinant and non-recombinant. 10. STEP 4. Cloning of cells (and foreign genes). Plate out the transformed bacteria on solid nutrient medium containing gene of interest and a sugar called X-gal. Only bacteria that have the gene of interest plasmid will grow. The X-gal in the medium is used to identify plasmids that carry foreign DNA. 11. STEP 5. Identifying cell clones with the right gene. In the final step, sort through the thousands of bacterial colonies to find those containing the cloned gene of interest. One technique, nucleic acid hybridization, depends on base pairing between the gene and a complementary sequence, a nucleic acid probe, on another nucleic acid molecule. 12. Recombinant DNA Technology: The Big Picture Copyright 2002 Pearson Education, Inc., publishing as Benjamin Cumm 13. Thank You

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