lec26 - Lectures 26, 27 and 28 recombinant DNA technology...

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Picture 90 Lectures 26, 27 and 28– recombinant DNA technology I. Goal of genetics A. historically - easy to isolate total DNA - difficult to isolate individual gene B. recombinant DNA technology C. why get the gene? 1. determine nucleotide sequence 2. comparison of gene sequences 3. comparison with proteins of known function 4. can manipulategene and reintroduce 5. introduce gene into different species D. overall strategy to clone a gene 1. isolate DNA from organism 2. clone fragments into vector 3. introduce into bacteria 4. find colony with clone of interest 5. use clone II. How to clone a gene, in greater detail: A. isolate DNA 1. genomic DNA - break cells open under conditions that stabilize DNA - can use whole organism, or specific tissue - extract the DNA 2. cDNA 1
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Picture 91 Picture 92 Picture 93 - isolate mRNA, reverse transcribe to generate cDNA B. break into fragments 1. why? chromosomes too large to work with 2. how?  a. by restriction endonucleases b. by random shearing C. clone into “vector” 1. why? 2. choose vector – many types available, some easier to work with, others accommodate  larger DNA pieces or offer other advantages a. plasmid – small, circular DNA i. replicates independent of chromosome 2
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Picture 94 ii. often many copies per cell iii. pUC – ampR, can accept small inserts
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lec26 - Lectures 26, 27 and 28 recombinant DNA technology...

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