Most of the techniques described in this chapter (blotting, cloning, PCR, etc.) are dependent on intermolecular attractions (annealing) between different populations of nucleic acids. Length of the strands, temperature, and percentage of GC nucleotides weigh considerably on intermolecular associations. Two other components commonly used in hybridization protocols are monovalent ions and formamide. A formula that takes monovalent ion (Na+) and formamide concentrations into consideration to compute a Tm (temperature of melting) is as follows:
Tm = 81.5 + 16.6(log M[Na+]) + 0.41(%GC) – 0.72(%formamide)
(a) For the following concentrations of Na+ and formamide, calculate the Tm. Assume 45% GC content.
Na+
% Formamide
0.825
20
0.825
40
0.165
20
0.165
40
(b) Given that formamide competes for hydrogen bond locations of nucleic acid bases and monovalent cations are attracted to the negative charges of nucleic acids, explain why the Tm varies as described in part (a).
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