Denaturation of DNA

Question 1

Denaturation

Answer 1

Complete unwinding and separation of the complementary single strand is called denaturation

Occurs only in vitro

Question 2

UV light

Answer 2

Absorption of UV light can be used to measure the extent of denaturation. Measurments are made at a wavelength of 260 nm – close to the absorbance maximum for nucleic acids.

Single stranded DNA absorb 12% to 40% more light than double stranded DNA.

Question 3

Melting Curve

Answer 3

A plot of change in absorbance of a DNA solution versus temperature.

The absorbance increase sharply at the melting point and the transition from double stranded to single stranded DNA takes place over a narrow range of temperature.

Question 4

Sigmoid shape of the curve

Answer 4

Denaturation is a cooperative process, as the rapid unzippering of the double stranded molecule, as the many hydrogen bonds and stacking interactions are disrupted.

Question 5

G/C vs. A/T

Answer 5

G/C denatures at a much higher temperature than A/T

It is easier to melt A/T rich DNA than G/C rich because A/T base pairs have weaker stacking interactions. And the stacking interactions are the first interactions to be disrupted by higher temperature.

**Easier to unwind localized regions of A/T base pairs

Question 6

Annealing

Answer 6

As the process by which two strands of complementary nucleic acid bases bind to each other. It is measured by the rate at which this occurs as it is a kinetic process. The rate is dependent upon the concentration of the strands in the solution, the length of these strands, and the uniqueness of the solutions

Question 7

Melting

Answer 7

The process by which two strands of complementary nucleic acid bases separate from each other. It is characterized by the temperature at which it is half completed (Tm) as it is a thermodynamic process. The melting temperature is dependent upon the percent composition of cytosine/guanine base pairs.