Lecture 25 W11 - Lecture 25 (3-16-11) Notes I. Cloning...

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Lecture 25 (3-16-11) Notes I. Cloning human growth hormone gene for mass production A. Human growth hormone (GH) is produced and secreted from the pituitary gland. 1. Low GH results in pituitary dwarfism 2. Can be treated with bovine GH, but this is expensive, because their isn’t that much GH protein present in the bloodstream at any given time. B. Researchers wanted to engineer E. coli to produce large amounts of GH. 1. they isolated mRNA from human pituitary gland tissue and converted it to DNA using an enzyme called reverse transcriptase (RT). RT is normally used by retroviruses and can synthesize DNA from an RNA template. 2. The DNA made from mRNA by RT is called complementary DNA (cDNA). Short DNA primers are then attached to both ends of each cDNA using DNA ligase. These primers contain a specific sequence that can be recognized and cleaved by a specific restriction enzyme. 3. Restriction enzymes are a form of primitive immune system in bacteria, but humans use these site-specific endonucleases to create new DNA molecules. 4. After cutting the cDNAs with a specific restriction enzyme, these cDNAs are ligated (covalently inserted) into a plasmid that was cut with the same restriction enzyme. C. Plasmids are circular DNA that must contain at least three things.
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Lecture 25 W11 - Lecture 25 (3-16-11) Notes I. Cloning...

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