DNA can undergo reversible denaturation, for example, by heat. a) Given the foll
ID: 223393 • Letter: D
Question
DNA can undergo reversible denaturation, for example, by heat. a) Given the following DNA denaturation curve, explain how can you measure the DNA denaturation process, explain the meaning of Tm, and how it can be affected by the base composition of the DNA (which base pairs will be more abundant in the DNA of the red curve?). b) The Polymerase Chain Reaction (PCR) is a procedure that uses the reversible DNA denaturation by heat, to amplify a DNA sequence. Explain briefly what is a polymerase and what is special about the DNA polymerase used for PCR, what is a primer, what would you need to mix in a tube for a PCR reaction, and what are the different steps that must take place during a PCR reaction.Explanation / Answer
Melting curve analysis is used to check the dissociation charecterstics of double stranded DNA on heating. Raise in temperature will dissociate the double strands and increases the absorbance leading to hyperchromicity. The temperature at which 50% of the DNA is denatured is the melting point. Tm is the melting temperature which is defined as the temperature at which half of the dsDNA molecules denature and are single stranded.
dsDNA with High G+C content will have a high Tm as there are 3 bonds between G and C, higher melting temperature(Tm) is required to denature or melt these bonds. On the other hand AT has only 2 bonds so low temperture (Tm) will be required
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