a. Ignoring the rules of optimal primer design, design TWO 20 base pair long polymerase chain reaction (PCR) primer sequences that satisfy all of the following criteria:
- Amplicon will encode only for the first 100 amino acids of the receptor;
- Amplicon will have custom restriction enzyme recognition sites: HindIII (AAGCTT) and BamHI (GGATCC) at the 5’ and 3’ ends respectively;
- Amplicon will have a mutation of D (GAT) in place of N (AAC) at amino acid position 4.
Your answer must include underlined restriction enzyme recognition sites and mutated sequences. [5 marks] (C6, CLO2, PLO2)
b. Based on the TWO primers designed in part a, analyse if the GC contents are considered optimal and justify why. Your answer must include the formula, step-by-step calculations and answers with units. [6 marks] (C4, CLO2, PLO2)
c. Based on the TWO primers designed in part a, determine the Tm of each primer. Your answer must include the formula, step-by-step calculations and answers with units. [5 marks] (C4, CLO2, PLO2)
d. Based on the Tm analysis in part b, determine the optimal annealing temperature for setting a thermal cycler. Your answer must include the formula, step-by-step calculations and answers with units. [4 marks] (C4, CLO2, PLO2)
e. If the given template from Figure 1 is in mRNA form instead, select a PCR variation to synthesize a DNA based equivalent. Your answer must include an elaboration of the key reagents and processes involved. [5 marks] (C4, CLO2, PLO2)