Genetics-Fall07(11-8-2007) - Recombinant DNA revolution:...

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Recombinant DNA revolution: Key discoveries that started this- the goal is to understand what cloning a gene means u want to isolate a gene and make lots and lots of copies of it how do u get that DNA/gene out of a cell and make those copes 1 way to make those copies is to put it a microorganism and if it make the copies or use mechanisms of…? What do we need to do to cut out a gene? 1) cut the DNA up into smaller pieces how? with nucleases (which are enzymes that cut the phospho-diester backbone of DNA we want nucleases that cut the DNA at specific sites (not at random) (so, at specific base sequences) so degrading foreign DNA would be good for an effective immune system to prevent infection the host immune system however, needs to be able to recognize ur own DNA vs. the foreigner invaders DNA (this is done by host/modification which occurs by methylation in restriction modification, ur DNA is methylated but the invaders isn’t and the enzymes will recognize the un-methylated and degrade it, but not urs (b/c its been methylated in the modification) this is done by restriction/modification enzymes these enzymes recognize palindromes (sequences that are the same whether they’re read forward or reverse) (they have names like EcoR1 which is E. coli restriction enzyme 1) so pple discovered that these enzymes will cut DNA that is NOT methylated at specific sites!! Bacteria also have something called R plasmids (resistance plasmids) (bacteria that are resistant to antibiotics have R plasmids) 2) we need to amplify DNA, and we want to let bacteria do this for us the R plasmid (circular piece of DNA) confers immunity to antibiotics these R plasmids therefore must carry info for an origin of replication, DNA methylation pattern (to prevent being attacked by the host bacteria immune system), a pattern for creation of resistance protein (proteins that alter the structure of DNA bacteria which confers immunity to antibiotics) (this is a selectable gene marker this gene confers a phenotype the phenotype is antibiotic resistance
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Genetics-Fall07(11-8-2007) - Recombinant DNA revolution:...

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