Hyperchromic shift on DNA melting

Hyperchromic shift on DNA melting

Aim

To  demonstrate  hyperchromic  shift  on  DNA  melting.

Principle

Hyperchromicity is the an  increase  in the absorbtion of ultraviolet light by a solution of DNA when subjected to heat, alkaline conditions, etc. Hyperchromicity of DNA occurs when the DNA duplex is denatured .The UV absorbance  is increased when the DNA strands are being separated, either by heat or by addition of  denaturant or by increasing  pH level. Heat denaturation of DNA is also called melting when DNA in solution is heated above its melting temperature [usually more than 80oC ], the double stranded DNA unwinds to form single stranded DNA. The bases becomes unstacked and can thus absorb more light. The G + C content often determined from the melting temperature [Tm] of DNA . In double stranded DNA ,three hydrogen bonds joins G C base pairs, and 2 hydrogen bonds connect AT bases pairs. DNA with a greater G + C content have more hydrogen bonds and its strands separate at higher temperature that it has a higher melting point.  DNA melting can be easily followed spectrophotometrically because the absorbance of DNA at 260 nm [UV light] increasing during strand separation.  When a DNA sample is slowly heated the absorbance increases as hydrogen bonds are broken single stranded.

Material Required

è DNA solution

è Waterbath              

è Thermometer

Procedure

è 5 ml of stranded DNA solution was pipetted out into 3 test tubes.

è One of the test tube was kept at room temperature.

è The other 2 test tubes were kept in a boiling waterbath for 10 minutes at 80oC  and 100oC  respectively.

Result

The optical density of the test tube samples  were found to be increased with  an increase in the temperature.

 


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