IntroductionEukaryotic genomes are much more complex than prokaryotic genomes. And further, plant genomes are more complex than other eukaryotic genomes. Prior to the development of recombinant DNA technology genomes, were analyzed by reassociation kinetics techniques. Reassociation kinetic experiments are performed by melting DNA and allowing it to reanneal upon itself or with another population of either DNA or RNA molecules. The kinetics of the reassociation provide data that can be used to analyze the overall structure, evolution and expression of genomes.
The most common method of denaturing duplex DNA is by heating to 100oC. But how can we monitor this denaturation and subsequent renaturation? The most common method is by measuring the absorbance change in the ultraviolet region at 260 nm. The important property is that melted, single-stranded DNA absorbs about 40% more at 260 nm than duplex DNA. If we slowly heat DNA the absorbance will increase dramatically over a short range of temperatures. The mid-point of this transition is called the melting temperature or Tm. Under physiological conditions, the Tm usually lies in the range of 85-95oC. Thus, without altering the cellular conditions, the duplex DNA is stable in the cell. The exact temperature that a particular DNA melts depends on several parameters. The GC content is important because GC base pairs have three hydrogen bonds compared to the two for AT base pairs. Thus, the higher the GC content the higher the Tm.
Effect of GC content on Tm
DNA denaturation is reversible, and the reversible process is called renaturation. This process requires that the temperature be lowered gradually. As this lowering of temperature proceeds the following occurs:
Hybridization - the pairing of complementary nucleic acids
Performing hybridization experiments in a solution is called liquid hybridization. We have already discussed the related procedure called filter hybridization.
Copyright © 1998. Phillip McClean