9.4.4—
Dissociation and Reassociation of DNA
When double-stranded DNA in solution is heated above a certain temperature the hydrogen bonds between complementary base pairs are broken and the two strands dissociate. The process, which can also be brought about by treatment of DNA with alkali, is commonly referred to as melting or denaturation. Strand separation is accompanied by an increase of approximately 35% in the absorbance of ultra-violet light. This hyperchromic effect provides a means of monitoring the dissociation of double-stranded DNA by measuring the increase in absorbance of a DNA solution at 260 nm as the temperature is raised. The melting curves of DNA from a variety of sources are compared in Fig. 9.6. The temperature at which 50% of the DNA is melted, the Tm, depends upon a number of factors such as the salt concentration and pH. Under standard conditions, i.e. when the DNA is dissolved in standard saline citrate (S.S.C. being 0.15 M sodium chloride, 0.015 M trisodium citrate, pH 7) the base composition and the Tm are related in the following way:
In viruses, the majority of sequences within the genome are similar in base composition and for a given DNA a sharp melting profile is observed with a characteristic Tm. In contrast, higher organism DNA often consists of distinct subfractions, each with a slightly different Tm. This produces a broad melting
Figure 9.6
Melting curves of various DNA samples. (1) Musk melon main-
band and (4) satellite DNA. (3) a 2:1 mixture of DNA from bacterium
C and phage P58. (2) B. subtilis DNA. Melting was carried out in S.S.C.
(From Bendich & Anderson, 1974.)
profile which represents a number of overlapping melting curves. With DNA samples from organisms that contain prominent satellite bands in caesium chloride the melting curve often has two or more phases. Even where no large satellites are present heterogeneity within the DNA can readily be detected by plotting the rate of change of absorbance at 260 nm of a DNA solution as a function of temperature.
If, following denaturation of DNA by alkali or by heating, the pH of the solution is restored to neutrality or the temperature reduced below the Tm, complementary strands of DNA show a marked tendency to recombine to form double-stranded molecules. This process of duplex formation is termed renaturation or reassociation. The rate of the reaction is governed by the concentration of DNA sequences, pH, temperature and salt concentration. Renaturation is normally carried out at moderately high salt concentrations at a pH close to neutrality and at a temperature approximately 25°C below the Tm. Under these conditions, single-stranded DNA molecules collide at random. Most collisions occur in such a way that complementary sequences of bases are not in register and the strands do not reassociate. If, however, two strands approach in an anti-parallel configuration and potential regions of complementarity are in close proximity a nucleation event will take place and hydrogen bonds will form. Whether or not the strands remain together depends upon the nature of the neighbouring bases on either side of the nucleation site. If complementary sequences are arranged in the correct register along the length of the molecule a rapid zippering effect takes place to produce a stable double-stranded DNA molecule.
DNA renaturation can be followed by measuring any property that distinguishes single-stranded from double-stranded DNA. The decrease in absorbance at 260 nm that accompanies duplex formation is often exploited for
this purpose but fractionation by hydroxyapatite is also commonly used. Under appropriate conditions only double-stranded DNA binds to hydroxyapatite (a hydrated form of calcium phosphate); single strands pass straight through a column and double-stranded material may subsequently be eluted either by increasing the salt concentration or by raising the temperature above the Tm. This procedure has the advantage that the properties of the single-stranded and renatured fractions may subsequently be studied separately.