Molecular Genetics

Complementary single strands of DNA will reassociate to form double-stranded DNA.

Facts

The absorbance of DNA at 260 nm (the absorption maximum) was followed as the DNA was slowly heated. The absorbance increased over a narrow range of temperature (left, upper). The mid point, Tm , of the transition to increased absorbance or hyperchromicity, depends on the base composition and sequence of the DNA. DNA heated to well above the Tm was cooled quickly. The absorbance was followed during a slow second heating. The absorbance decreased (hypochromicity) (left, lower) and then increased. The hypochromicity was maximal about 25 C below the Tm. E. coli and T even phage DNA, behaved this way, but DNA from calf thymus failed to exhibit signfiicant hypochromicity.

Denatured DNA from deuterium oxide-grown E.coli cells (density labeled) was mixed with denatured DNA from normally grown E.coli cells and incubated at a temperature 25 C below the Tm. The resulting DNA was analyzed by CsCl density gradient centrifugation. In the diagram at right, L and H represent the positions of native DNA from water and deuterium oxide grown cells, respectively. The order of Tm's of the intermediate peak formed between dense E.coli DNA and DNA from other bacteria was: E.coli + E.coli>E.coli + Shigella>E.coli + Salmonella

Interpretations

• The two strands of the double helix are held together by weak non-covalent forces and can be dissociated by the input of thermal energy.
• The strands can reassociate by incubation at temperatures slightly below the Tm.
• Double-stranded DNA absorbs less 260 nm radiation than single-stranded DNA.
• Calf thymus DNA is more complex than E.coli DNA and thus renatures less extensively in the length of time allowed for renaturation.

• The middle peak in the density gradient is of intermediate density and must consist of one light strand and one heavy strand. It is a hybrid duplex. This experiment was the first proof that DNA renaturation could be observed.
• DNAs from related organisms have sequences sufficiently similar that their strands can hybridize with one another.
• Mismatches in a DNA duplex result in a lowering of the T_m.
• The T_m's of DNA hybrids can serve as a measure of the relatedness of organisms.

Further information

• Denaturation profiles and Tm's can be calculated from DNA sequence information.
• The DNA analyzed in these and other renaturation experiments consists of small pieces of the original DNA. Pieces were broken by shearing due to experimental manipulation. Intact chromosomes have a single long DNA molecule.
• Studies of the kinetics of reassociation provide a picture of the organization of complex genomes.

This is page 112281 of Molecular Genetics by Ulrich Melcher, © 1997- 2000