Thursday, 8 April 2021

Denaturation(Melting Curve) and Renaturation of DNA


   DNA is a double-stranded molecule. It is stabilized by chemical interactions. Most important of these interactions is the hydrogen bonds.

   In DNA double helix structure adenine(A) always pairs with thymine(T) via 2 hydrogen bonds, cysteine(C) always pairs with guanine(G) via 3 hydrogen bonds.

   Inside living cells the two strands of DNA separate from each other during DNA replication. This separation disrupts the hydrogen bonds between the bases and this task is done by proteins called helicases.

    In the laboratory the hydrogen bonds of DNA double helix can be disrupted by two methods -
First method is by changing pH of the DNA solution and
second method is by heating the DNA solution.

Denaturation (melting)

   DNA solution is heated as a result hydrogen bonds are disrupted and the double-stranded DNA separates into single strands. This separation of DNA strands is known as Denaturation or melting. 

  Thus denaturation of DNA is the loss of helical structure of DNA as the temperature increases, the percentage of DNA denaturation also increases. This can be shown graphically and the curve obtained is known as the melting curve of DNA.


Measurement of DNA Denaturation

   DNA denaturation is measured by using spectrophotometer. This is based on the fact that all nucleotide bases consist of aromatic rings. These aromatic rings absorb light in the ultraviolet(UV) range. Nucleic acids such as DNA are made up of these bases, therefore DNA molecule will also absorb light in the UV range. 

  All bases of DNA that is Adenine, Guanine, Cytosine and Thymine have a strong absorbance at 260 nm. Double stranded DNA absorbs less light at 260 nm compared to single stranded DNA. This is because base stacking interactions in DNA double helix interferes with the absorbance. 

  So as DNA denatures are melts its absorbance increases. Thus absorbance of a DNA solution is measured under particular set of conditions. Sigmoid shape curve obtained by plotting the absorbance values with increase in temperature results in the melting curve of DNA.

Melting Curve of DNA
   Lower portion of the melting curve represents that all the DNA molecules in the given DNA solution are intact. That is they are double-stranded and, Upper portion of the melting curve represents that all the DNA molecules have completely denatured or melted. 

  So as per figure we can see that as temperature increases DNA molecule melt and the two strands separate. Also as DNA becomes single-stranded its absorbance increases. 

  The temperature at which half of the DNA molecules are denatured is called as the melting temperature of DNA. That means at this temperature half of the DNA molecules present in the solution will be single-stranded and other half will be double-stranded. melting temperature is found at the midpoint of the melting curve besides denaturation.

DNA Renaturation

  Another feature of DNA double helix is that the separated complementary strands of DNA can spontaneously reassociate to form a double helix. This happens when the temperature of DNA solution is lowered below its melting temperature this phenomenon is known as Renaturation or annealing. 

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