Chapter_20_DNA_Technology

Chapter_20_DNA_Technology - CLONING GENES WITH RECOMBINANT...

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Step 1 Isolate the gene you wish to clone Isolate the plasmid from bacterial cells, usually E. coli. (important genes in plasmid includes amp R and lacZ ) Step 2- Inserting the gene into the plasmid The same restriction enzymes are used on both the gene and the plasmid to cut the plasmid and gene of interest at its specified position, leaving a single stranded “sticky end” on both ends. The two ends are complementary and join together, while DNA ligase joins the molecules to a recombinant DNA molecule. Keep in mind that this process is not 100% foolproof, and many other combinations of different genes are formed. Step 3- Reintroducing the vector into cells Through the bacterial cell’s transformation (uptake of naked DNA), bacterial cells once again take up its plasmids, this time with the foreign gene. Step 4- Replication and Cloning Bacteria placed in plate with nutrient medium, ampicillin, and the sugar X-gal. As bacteria divides and forms colonies, the foreign gene is also cloned. Only bacteria with plasmids (which contain the amp R gene resistant to ampicllin) can survive and plasmids with foreign DNA appear white. Step 5- Finding the Gene of Interest Recall that, besides the gene of interest, other animal DNA is also cloned. Bacterial colonies, which may or my not contain the gene we are looking for, are pressed to a special filter paper. The filter paper denatures the DNA into single strands that stick to the paper. If we know a part of the nucleotide sequence of our gene of interest, we can synthesize a complementary RNA or DNA, which is called the nucleic acid probe. Through the process of nucleic acid hybridization , the probe binds itself to the cloned DNA, which is now single stranded from the filter paper. The probe is tagged with a radioactive isotope, which allows us to easily find our gene of interest. By finding our desired gene, we can easily grow large numbers of it in liquid culture. DNA Technology is extremely relevant and applicable
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This note was uploaded on 04/01/2012 for the course BIO 141 taught by Professor Dr.cafferty during the Spring '11 term at Emory.

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Chapter_20_DNA_Technology - CLONING GENES WITH RECOMBINANT...

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