encoded by a gene and to determine its levels of transcription and tissue specificity. Methods exist for both
of these purposes and both depend upon the ability of a single-stranded nucleic acid to pair specifically
with its complementary strand.
5.5.1 Parameters that Affect Nucleic Acid Hybridisation
Hybridisation is the annealing of two single strands of a nucleic acid to form a duplex. Duplex strength is
measured by observation of the melting temperature(Tm) and is affected by several parameters.
5.5.1.1 Base Composition (%GC). Since G–C base pairs have three hydrogen bonds, they are
stronger than A–T base pairs, which have only two. Thus duplexes with higher G–C content have higher
melting temperatures.
5.5.1.2 Temperature (T). The rate of association of single-stranded DNA into a duplex varies
markedly with temperature (Figure 5.9: see also Section 2.5.1). The shape of this curve is governed by two
Nucleic Acids in Biotechnology 179
Figure 5.8 Gene synthesis by recursive PCR. Bars represent oligonucleotides and their extension products
after PCR