BCH 2333 Lecture Notes - Lecture 21: Low Copy Number, Recombinant Dna, Endonuclease
16 Dec 2017
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Molecular cloning (aka cloning: dna is copied exactly, done inside an organism by dna replication, need an extrachromosomal element (dna outside of the genome that can replicated independently) Polymerase chain reaction (pcr: enzymatic reaction - no need for organisms. Endonucleases cut both strands of dna: can leave sticky end 5" overhang (aka cohesive ends) or blunt ends, stick ends can associate by base pairing with other fragments generated by the same restriction enzyme. Very sequence selective - if not they would kill the cell that makes them. Recognize palindromic sequences (same in the forward and reverse direction - same when read 5" to 3" on both strands) Ligate dna into an extrachromosomal element - usually a plasmid, allowing dna to be replicated in a cell. Ligase uses atp to connect dna fragments - like in dna replication. Plasmids: circular dna duplexes of 1 - 200 kbp. Counting necessary genetic machinery (replication origin) necessary for autonomous replication.
