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Plasmid Maps = Restriction Maps Cloning Various meanings - copy Molecular biology – copy & paste DNA from 1 location to another (plasmid) Restriction Digest – CUT DNA Restriction endonucleases aka - Restriction Enzymes •Sequence specific •Cut in a particular pattern •Often leaves overhangs Restriction Site •Usually palindromic 5’ – GCACGAATTCCGGGGTTAC – 3’ 3’ – CGTGCTTAAGGCCCCAATG – 5’ 5’ – GCACGAATTCCGGGGTTA – 3’ 3’ – CGTGCTTAAGGCCCCAAT – 5’ EcoRI site DNA sequence Digested / cut with The EcoRI enzyme 5’ – GCACG AATTCCGGGGTTAC – 3’ 3’ – CGTGCTTAA GGCCCCAATG – 5’ Note overhangs What is left P O O P A-A-T-T-C-C-G–3’ G-G-C–5’ OH 5’-A-C-G OH 3’-T-G-C—T-T-A-A Overhangs allow them to reattach 5’-A-C-G 3’-T-G-C—T-T-A-A A-A-T-T-C-C-G–3’ G-G-C–5’ A-A-T-T-C-C-G–3’ 5’-A-C-G G-G-C–5’ 3’-T-G-C-T-T-A-A Ligase Connects DNA = Paste PO OH A-A-T-T-C-C-G–3’ 5’-A-C-G G-G-C–5’ 3’-T-G-C-T-T-A-A OH O P 5’ – GCACGAATTCCGGGGTTA – 3’ 3’ – CGTGCTTAAGGCCCCAAT – 5’ Cloning EcoRI site GAATTC CTTAAG Ori AmpR gene EcoRI enzyme EcoRI site Cut ends are still EcoRI G CTTAA AATTC G EcoRI site Ori AmpR gene Cloning EcoRI Human DNA EcoRI Base pairing G GAATTC CTTAA CTTAAG INSERT AATTC GAATTC G CTTAAG Ori AmpR gene 1. Fragmentation - Restriction endonuclease digest 2. Ligate (ligase) into plasmid vector Ligase to connect Plasmid Maps = Restriction Maps Perpindicular lines denote restriction sites Arrows represent genes and their directions Cloning EcoRI EcoRI GAATTC CTTAAG Human DNA VECTOR Ori AmpR gene EcoRI Base pairing GAATTC CTTAAG GAATTC CTTAAG INSERT Ori AmpR gene 1. 2. 3. 4. Fragmentation - Restriction endonuclease digest Ligate (ligase) into plasmid vector Transform – to replicate DNA Selection - grow on antibiotics Ligase to connect Transform
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