MCB 2050 Lecture Notes - Lecture 4: Pyrococcus Furiosus, Thermus Aquaticus, Klenow Fragment

• School: University of Guelph
• Department: Molecular and Cellular Biology
• Course: MCB 2050
• Professor: Dick Mosser

Document Summary

**techniques of molecular genetics (snustad 6e: ch 14)** Original protocol used klenow fragment of e. coli dna polymerase i: optimum activity is 37 c, needed to be added fresh after each cycle (heat-inactivated during denaturation step, taq dna polymerase: heat-stable dna polymerase made pcr procedure practical. Isolated from pyrococcus furiosus (from geothermal vents: very low error rate, best choice for gene cloning. Efficiency: % conversion of template to product per cycle. Error rate: frequency of errors per base pairs incorporated (cid:1007)" to 5" e(cid:454)o: e(cid:454)o(cid:374)u(cid:272)lease p(cid:396)oof(cid:396)eadi(cid:374)g a(cid:272)ti(cid:448)it(cid:455) Reaction mixture: target dsdna (double stranded dna, pair of oligonucleotide primers, thermostable dna polymerase, dntps. Using pcr to amplify dna molecules in vitro: specificity of pcr product defined by primers. Anneals to the bottom strand (has the same sequence as the top strand) Exponential increase in target dna copies after the third cycle. Must have the gene sequence in order to design gene specific primers.