You are given two solutions containing different purified DNAs. One is from the
ID: 973498 • Letter: Y
Question
You are given two solutions containing different purified DNAs. One is from the bacterium P. aeruqinosa and has a GC composition of 68%, whereas the other is from a mammal and has a GC composition of 40%. You measure the absorbance of light of each solution as a function of increasing temperature. Which solution has the higher Tm and why? After melting the two solutions, mixing them together, and allowing them to cool, what would you expect to happen? Would appreciable amounts of the bacterial DNA be found associated in a helix with mammalian DNA? Explain. Ethidium bromide is a fluorescent molecule that interacts with DNA and is widely used in electrophoresis. Based on the structure of ethidium bromide, how does if interact with the DNA molecule and what effect would this interaction have on the melting temperature, Tm of the DNA. Justify your answer.
Explanation / Answer
The percentage of GC content of DNA has a significant effect on its Tm. Because G-C pairs form three hydrogen bonds, while A-T pairs form only two, the higher the percentage of GC content, the higher its Tm.
DNA gets denatured upon heating and break into single-stranded state. While upon mixing of two different denatured DNA after cooling, they undergo hybridization.
Yes, bacterial DNA and mammalian DNA undergo hybridization and associate with each other. Hybrid molecules are formed between similar sequences and any differences between those sequences will result in a disruption of the base-pairing. On a genomic scale, the method has been used by researchers to estimate the genetic distance between two species, a process known as DNA-DNA hybridization.
EtBr binds strongly to the double strand DNA molecules. The EtBr being a planar molecule inserts itself perpendicularly to the axis of the helices, between sugar and the nitrogenous bases and interact with the base pairs through van der Waal's interactions. This becomes a tight bond because the water molecules present in the vicinity were displaced by the bulky Ethidium ions. Tm is the melting temperature at which 50% of DNA gets denatured. While upon binding the DNA with EtBr, the denaturation of DNA become difficult and consequently Tm of the DNA become increased.
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