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MCB 121 Lecture 5

MCB 121 Lecture 5 - MCB 121 Lecture 5 Midterm I Outline...

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MCB 121 - Lecture 5 Midterm I - 01/26/10 Outline: Recombinant DNA Cloning (Transformation) Molecular Cloning ------------------------------------------------------------------------------------------------------------------------- Definition of cloning: an identical genetic replica ex. E. coli colony- all the cells in the colony are the same ex. Vegetative plate propagation - piece of tree branch and plant it ex. Molecular cloning: making specific copy of single piece of DNA Molecular cloning: - depends on recombinant DNA (move piece of DNA around to get them replicate appropriately - invented in early 70's by Stan Cohen + Herbert Boyer - three things brought together for molecular cloning: 1. restriction enzymes 2. DNA ligase 3. Plasmids Restriction enzymes : - also called restriction endonucleases - endonucleases: enzyme cut nucleic acid in the middle of some place - restrict the DNA being transferred - a defensive mechanism against viruses - can be used for other purposes * it makes double stranded cuts at specific DNA sequence ex. Eco. RI => cut at sequence - G*AATTC- between G and A ** this sequence has "palindrome property" => the sequence 5' to 3' on one strand is the same on another strand from 5' to 3' -----------G*AATT C ... C TTAA*G------------- ** After cut: only 4bp still pairing together, doesn't provide sufficient energy to keep them together even at room T. These two regions would fall apart into 2 fragments. -----------G OH3' 5'P A A T T C ---------- -----------C T T A A P5' 3'HO G----------
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<= sticky ends (stick and fall apart on and off) <= results 5' overhangs (the end that's hanging over is the 5' end) ** Depends on where the cut is made, it can be either 3' or 5' overhang or even blunt end. ex. Sma I => --------------CCC*GGG-------------- ---------------GGG*CCC-------------- <= blunt ends , no sticky ends * key to restriction enzyme: produce identical fragments from a solution of identical DNA molecules DNA ligase: - in DNA replication, DNA ligase use ATP energy to link two Okazaki fragments together * repairing the "nick"; all the bases are paired, but a place has P-S backbone with a break - in sticky ends: DNA ligase repairs gaps -------------G .. A-A- T- T - . C------------- -------------C - T- T- A- A G-------------- - in recombinant DNA: T4 ligase is often used * it even ligates blunt ends - for ligase to work: 5'P and 3'OH are required with energy from ATP
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MCB 121 Lecture 5 - MCB 121 Lecture 5 Midterm I Outline...

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