Chapter 20, Problem 31ESP

Most of the techniques described in this chapter (blotting, cloning, PCR, etc.) are dependent on hybridization (annealing) between different populations of nucleic acids. Length of the strands, temperature, and percentage of GC nucleotides weigh considerably on hybridization. Two other components commonly used in hybridization protocols are monovalent ions and formamide. A formula that takes monovalent Na⁺ ions ((M[Na⁺)) and formamide concentrations into consideration to compute a T_m (temperature of melting) is as follows:

T_m = 81.5 + 16.6(logM[Na⁺]) + 0.41(%GC) − 0.72(%formamide)

1. (a) For the following concentrations of Na⁺ and formamide, calculate the T_m. Assume 45% GC content.

1. (b) Given that formamide competes for hydrogen bond locations on nucleic acid bases and monovalent cations are attracted to the negative charges on nucleic acids, explain why the T_m varies as described in part (a).