Monitoring of DNA denaturation in vitro is done by ?
Ans. is 'b' i.e., UV absorptionDenaturation (melting) of DNADenaturation (melting) refers to separation of the two strands of the DNA into two component strands. When temperature of medium is raised, the hydrogen bonds linking of complementary base pair tends to break. As a result, separation of two strands occur. Melting temperature (Tm) indicates the temperature at which half of the double stranded structure is lost. Normally, Tm is between 85deg C and 95deg C.Since G and C are linked more strongly by three hydrogen bonds, in contrast to two hydrogen bonds linking A and T, G and C linking provides thermostability to DNA. Therefore, DNA molecules having higher GC content have relatively higher Tm. For every 10% increase in GC content, melting temperature increases by 5degC. Simple formula is -Tm (degC ) = 2 (number of AT base pairs) + 4 (number of GC base pairs)In addition to heat, alkali treatment and decrease of salt conentration also cause denatruation. Decrease in salt concentration decreases Tm. Therefore, increase in salt concentration increases DNA duplex stability. Denaturation affects propeies of DNA and these can be used to monitor strand separation -i) Hyperchomicity (hyperchromic effect) : DNA bases (purines and pyrimidines) absorb UV light of 260 nm. Upon denaturation, this optical absorbance is increased and is called hyperchromicity of denaturation or hyperchromic effect.ii) Viscosity : Viscosity of the DNA solution decreases on melting (denaturation) because single strands are more flexible than the stiff, resilient double helix.The separated strands of denatured DNA can rejoin on gradual cooling. This is called renaturation or reannealing.
Centrifugation
UV absorption
Chromatography
HPLC