Melting temperature (Tm)

(Melting temp.-Tm)

The temp. at which half the strands of DNA are single stranded and half are double stranded. Half of the DNA are denatured . (Tm) It  is characteristics of Dna compostion ( GC content) .The higher the G+C content higher the Tm. 

                                               

                                                           

Image source-http://cbc.arizona.edu/classes/bioc461/Chapter5Part1Notes.htm

Image source-http://www.biology.arizona.edu/biomath/tutorials/linear/linearfunctionapplication/dnamelt.html

            

                      Tm directly proportional to G+C content 

Q.What is the maximal t_m for a DNA sample that is 500.0 bp long?

Tm is useful for monitoring your PCR reaction because it lets you distinguish between specific and unspecific amplification. Of course it is easy to see if you have dimer formation if you run your PCR products on a gel, but

with many samples it soon becomes tedious.

You can monitor DNA melting through UV light. The aromatic bases in DNA absorbs UV light (260nm). But when the two strands are hydrogen bonded with each other, the UV light is not absorbed readily. However, when they melt, the bases are exposed and the absorption goes really high. So if you shine UV light onto your sample and raise the temperature of your sample, you will see a sudden increase in absorbency when the DNA melts.

The Tm can be determined experimentally or calculated from simple formula -

   

    Tm=(4x [G+C]) + 2x[A+T]) 

Factirs that affect the Tm -
1.Concentration of DNA.
2.Concentration of ions in the solution ,most notably Mg2+ and K+.
3.DNA sequence
4.Length of Dna.

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