PHYSICS 102 Lecture Notes - Lecture 19: Thermus Aquaticus, Kary Mullis, De Novo Synthesis
Document Summary
History of pcr: conceived in 1980s by kary mullis while at cetus, used normal" dna pol but given boost by discovery of. Thermostable dna polymerases: 2 categories; thermophilic and hyperthermophilic, most famous; taq (thermus aquaticus, taq; ~94kda, 832 aa protein, 2 domain structure; n-terminal contains 5"-3", optimal reaction temp. Dna strands: mixture incubated with dna pol and 4 dntps so dna can be synthesised starting from 2 primers. Pcr components: nucleotides must be dntps, conc. Linear dna may be better than circular: buffer usually tris-based, ph 8. 3 - 8. 8 @ rt, 50mm kcl usually included although increasing to 70 - 100mm may help with small amplicons: taq produce a-tailed dna molecules, pfu and other proof-readers yield blunt-ended. Cloning pcr products: depends on polymerase used for amplification, restriction sites added to primers, for products amplified by non-proofreaders, ligate into purchased or constructed t-vector", for products produced using restriction enzyme-
