Worksheet 1. Look at each PCR component listed below. For each one, determine which steps(s) of the PCR reaction (denaturation, annealing or extension) would be directly affect if that component were missing. a. Taq polymerase: Extension b. Oligonucleotide primers: Annealing c. DNA template: Denaturation d. Deoxynucleotides (A, T, G and C): Extension 2. Imagine that you correctly prepare your PCR reaction mixture, but there is something wrong with the thermal cycler. Describe what would happen if: a. The thermal cycler was stuck on 940C: Denaturation take place, but primers can’t anneal, therefore no annealing or extension. b. The thermal cycler cycled between 600C and 720C, but never reached 940C No Denaturation, therefore primers can’t anneal so no annealing or extension c. The thermal cycler cycled between 940C and 720C, but never reached 600C Denaturation takes place, but primers can’t anneal, therefore no annealing or extension 3. Look at the double-stranded segment of DNA shown below. Imagine that the two strands have already been denatured, and the temperature has been decreased to an appropriate annealing temperature. Show where the two primers would anneal to the strands, then indicate the direction of extension on each new strand with an arrow. 5’--T C A G G A C G T A A G C T T G C A T A T C T C G A T G C T A A A T C A T—3’ 3’ A C G A T T T 5’ 3’--A G T C C T G C A T T C G A A C G T A T A G A G C T A C G A T T T A G T A—5’ 5’ G G A C G T A 3’ Primer #1: 3’ A C G A T T T 5’ Primer #2: 5’ G G A C G T A 3’