lecture 25 W11 - Lecture 25 Recombinant DNA Today's topics...

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Today’s topics 1) Describe how the gene for human growth hormone was cloned in a bacterial plasmid 2) Explain how the polymerase chain reaction (PCR) works 3) Explain the basics of DNA sequencing Lecture 25 (3/16/11) - Recombinant DNA
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Molecules; Chemical bonds; Free energy Lipids Amino acids nucleotides carbohydrates membranes proteins Membrane transport Membrane proteins cytoskeleton enzymes DNA RNA Metabolism Sugar transport Electrolyte transport Kidney Nerve cells Muscle & movement Transporters and pumps Receptors Cell-cell communication Chemical signaling development Replication transcription translation Glycolysis Fermentation Krebs cycle Photo- Synthesis & Calvin cycle Cell cycle Regulation of transcription biotechnology genomics Microbes & viruses Biology 172 flowchart (lecture 25) cancer epigenetics
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(a) GH1 codes for a pituitary growth hormone. (b) Normal versus GH1-deficient Normal GH1 gene Defective GH1 gene Little or no GH1 protein produced in pituitary gland Pituitary dwarfism (slower growth, shorter stature) Normal amount of GH1 protein produced
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1) A secreted signaling molecule that binds to specific cell surface receptors, leading to activation of a cytosolic kinase 2) GH is synthesized in the pituitary gland 3) GH can promote cell growth and division in many cell types 4) Individuals with defective GH synthesis suffer from pituitary dwarfism . 5) In the 1980’s there was considerable interest in identifying the GH and insulin genes 6) How to do this??? Growth hormone (GH)
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Reverse transcriptase Single-stranded cDNA mRNA or Double-stranded cDNA Reverse transcriptase Double-stranded cDNA DNA polymerase Primer Reverse transcriptase - an enzyme used by retroviruses which uses RNA as a template for DNA synthesis. Reverse transcriptase can Convert an mRNA to complementary DNA (cDNA). The single Stranded cDNA can be converted to double stranded cDNA by Reverse transcriptase or DNA polymerase. Use reverse transcriptase to create a cDNA library from pituitary gland mRNA. Why use pituatary gland as the source of mRNA?
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After cDNA synthesis, short DNA sequences containing a restriction enzyme site are attached to each cDNA. This allows the cDNA to be inserted into a cloning plasmid.
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