Info

depth

very deep

Giycosyl-bond conformation

anti

anti

anti at C syn at G

Source Adapted from DiCkeison R E etal. 1362. CSHSQB47: 14. Copyright© 1982 Colei Spring Harbor Laboratory Press. Used with permission

Source Adapted from DiCkeison R E etal. 1362. CSHSQB47: 14. Copyright© 1982 Colei Spring Harbor Laboratory Press. Used with permission

FIGURE 6-14 Reannealin g and hybridization. A mixture of two Otherwise identical double-stranded DMA molecules, one normal wild-type DNA and the otfier a mutant missing a short stretch of nucleotides (marked as region a in red), are denatured by heating. The denatured DNA molecules are allowed to renature by incubation |ust below the melting temperature. This treatment results in two types of renatured molecules One type is composed of completely renatured molecules in \Miich two complementary wild-type strands reform e helix and two complementary mutant strands reform a helix. The other type are ftybrid molecules, composed of a wild-type and a mutant strand, exhibiting a start unpaired loop of DNA (region a)

FIGURE 6-14 Reannealin g and hybridization. A mixture of two Otherwise identical double-stranded DMA molecules, one normal wild-type DNA and the otfier a mutant missing a short stretch of nucleotides (marked as region a in red), are denatured by heating. The denatured DNA molecules are allowed to renature by incubation |ust below the melting temperature. This treatment results in two types of renatured molecules One type is composed of completely renatured molecules in \Miich two complementary wild-type strands reform e helix and two complementary mutant strands reform a helix. The other type are ftybrid molecules, composed of a wild-type and a mutant strand, exhibiting a start unpaired loop of DNA (region a)

for several indispensable techniques in molecular biology, such as Southern blot hybridization (see Chapter 20) and DNA microarray analysis (see Chapter 18, Box 18-1).

important insights into the properties of the double helix were obtained from classic experiments carried out in the 1950s in which the denaturation of DNA was studied under a variety of conditions. In these experiments, DNA denaturation was monitored by measuring the absorbance of ultraviolet light passed through a solution of DNA. DNA

maximally absorbs ultraviolet light at a wavelength of about 2b0 nm. It is the bases that are principally responsible for this absorption. When the temperature of a solution of DNA is raised to near the boiling point of water, the optical density, called absnrbaiice, at 260 nni markedly increases, a phenomenon known as hyperchromicity. The explanation for this increase is that duplex DNA absorbs less ultraviolet light by about 40% than do individual DNA chains. This hypochromicity is due to base stacking, which diminishes the capacity of the bases in duplex DNA to absorb ultraviolet light.

If we plot the optical density of DNA as a function of temperature, we observe that the increase in absorption occurs abruptly over a relatively narrow temperature range. The midpoint of this transition is the melting point or Tm (Figure 6-15}. Like ice, DNA melts; it undergoes a transition from a highly ordered double-helical structure to a much less ordered structure of individual strands. The sharpness of the increase in absorbance at the melting temperature tells us that the denaturation and renaturation of complementary DNA strands is a highly cooperative, zippering-like process, Renaturation, for example, probably occurs by means of a slow nudeation process in which a relatively small stretch of bases on one strand find and pair with their complement un the complementary strand (middle panel of Figure 6-14). The remainder of the two strands then rapidly zipper-up from the nucleation site to reform an extended double helix (lower panel of Figure 6-14).

The melting temperature of DNA is a characteristic of each DNA that is largely determined by the G:C content of the DNA and the ionic strength of the solution. The higher the percent of G:C base pairs in the DNA (and hence the lower the content of A:T base pairs), the higher the melting point (Figure 6-16). Likewise, the higher the salt concentration of the solution, the greater the temperature at which the DNA denatures. Howr do we explain this behavior? (j:C base pairs contribute more to the stability of DNA than do A:T base pairs because of the greater number of hydrogen bonds for the former (three in a GrC base pair versus two for A:T) hut also importantly, because the stacking interactions of G:C base pairs with adjacent base pairs are more favorable than the corresponding interactions of A:T base pairs with their neighboring base pairs. The effect of ionic strength reflects another fundamental feature of the double helix. The backbones of the two DNA strands contain phosphoryl

FIGURE 6-15 DNAdenaturation curve.

single stranded

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