LEC22 - Recombinant DNA technology and Gene cloning Early...

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Recombinant DNA technology and Gene cloning
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Early studies of lambda led the way to recombinant DNA technology
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Cos sites are 12 bases long pGGGCGGCGACCTC G G CCCCGCCGCTGGAp 100,000 BP 5’ 3’ CCCCGCCGCTGGAG GGGGCGGCGACCTC
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Could cohesive ends allow one to bring two different DNAs together? How could cohesive ends be made? Restriction/modification system was discovered again from bacteriophage studies.
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Observation: Phage grown on a particular strain of E. coli , could infect same strain and give high yields of progeny High yield (100’s phage)
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Phage grown in different strain of E. coli would give low yield of progeny Low yield (no phage)
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Restriction/Modification Phage DNA was cleaved into fragments
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Host DNA was protected from cleavage by modifying DNA with methylation Not cleaved by Restriction enzymes
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If phage DNA was methylated at correct sequences, the DNA was protected from restriction cutting, yielding high numbers of progeny High yield (100’s phage)
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If DNA was not methylated at the correct sequences, the DNA was cut with restriction enzyme and very few progeny were formed Low yield (no phage)
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Restriction/modification system Primitive immune system Protected bacteria from viruses Each strain of bacteria had different recognition sequence (different restriction enzyme), but always modified DNA at same sequence as restricted
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Restriction Enzymes Sequence specific endonucleases (not exo) Each bacterial organism has its own specific restriction system
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