nucleation is rate-limiting at low concentrations and each duplex zips to completion almost instantly
( 1000 bp s^1 ). The nucleation process is bi-molecular, so renaturation is concentration dependent with a
rate constant around 10^6 M^1 s^1.^66 It is also dependent on the complexity of the single strands. Thus, for
the simplest cases of homo-polymers and of short heterogeneous oligonucleotides, nucleation sites will
usually be fully extended by rapid zipping-up. This gives us an ‘all-or-none’ model for duplex formation.
By contrast, for bacterial DNA each nucleation sequence is present only in very low concentration and the
process of finding its correct complement will be slow. Lastly, in the case of eukaryotic DNA the existence
of repeated sequences means that locally viable nucleation sites will form and can be propagated to give
relatively stable structures. These will not usually have the two strands in their correct overall register.
Because such pairings become more stable as the temperature falls, complete renaturation may take an
infinitely long time.
Longer nucleic acid strands are able to generate intra-strand hairpin loops, which optimally have about
six bases in the loop and paired sections of variable length. They are formed by rapid, uni-molecular
processes which can be 100 times faster than the corresponding bimolecular pairing process. Although
such hairpins are thermodynamically less stable than a correctly paired duplex, their existence retards the
rate of renaturation, so that propagation of the duplex is now the rate-limiting process (Figure 2.44). One
notable manifestation of this phenomenon is seen when a hot solution of melted DNA is quickly quenched
to4°C to give stable denatured DNA.
With longer DNA species, Britten and Kohne have shown that the rate of recombination, which is moni-
tored by UV hypochromicity, can be used to estimate the size of DNA in a homogenous sample. The time
tfor renaturation at a given temperature for DNA of single-strand concentration Cand total concentration
C 0 is related to the rate constant kfor the process by an equation which in its simplest form is:
C/C 0 (1 kC 0 t)^1In practice where C/C 0 is 0.5 the value of C 0 tis closely related to the complexity of the DNA under
investigation.
This annealingof two complementary strands has found many applications. For DNA oligomers, it pro-
vided a key component of Khorana’s chemical synthesis of a gene (Section 5.4.1). It is now an integral
feature of the insertion of chemically synthesised DNA into vectors. For RNADNA duplexes, it has pro-
vided a tool of fundamental importance for gene identification (Section 5.5) and is being explored in the
applications of antisense DNA (Section 5.7.1).
2.5.2 DNA Breathing
Complete separation of two nucleic acid strands in the melting process is a relatively slow, long-range
process that is not easily reversible. By contrast, the hydrogen bonds between base pairs can be disrupted
66 Chapter 2
Table 2.9 Thermal stability matrix for nearest-neighbour stacking
in base-paired dinucleotide fragments with B-DNA
geometry
3 -Neighbour
5 -Neighbour A C G T
A 54.50 97.73 58.42 57.02
C 54.71 85.97 72.55 58.42
G 86.44 136.12 85.97 97.73
T 36.73 86.44 54.71 54.50
Numbers give Tmvalues in °C at 19.5 mM Na.