Question

A number of factors influence the melting properties of a linear, double-stranded DNA molecule in a 0.25M sodium chloride solution. Considering this, explain each of the following observations:

(a) The Tmincreases in proportion to the length of the molecule

(b) As the concentration of sodium chloride is decreased, the Tm decreases

(c) Renaturation of single strands to form double strands occurs more rapidly when the DNA concentration is increased

(d) The Tm value is reduced when urea is added to the solution

(e) The Tm value is reduced when cytosine bases undergo deamination

Answer 1

The Tm (melting temperature) of a DNA molecule is the temperature in which half the DNA molecules are denatured.

It depends on certain factors:

1. The Tm increases in proportion to the length of the molecule: Longer the DNA molecule, more H- bonds are there to hold the two strands together. And hence, more H- bonds must be broken for the two strands to separate.
2. As the concentration of sodium chloride is decreased, the TM decreases: Na⁺ shields the negative charges of the sugar – phosphate backbone from interacting with each other. The repulsion between the negatively charged phosphate backbones is the important force destabilizing the double helix DNA. Hence, increasing NaCl concentration increases the DNA helix stability i.e increases Tm and decreasing NaCl concentration decreases DNA helix stability.
3. Renaturation of single strands to form double strands occurs more rapidly when DNA concentration is increased: More DNA concentration means there a more chances for a DNA molecule to find complementary strand and therefore they will renature faster. On the other hand DNA molecule at low concentration will take more time to find the complementary strand and hence renaturation will be slower.
4. The Tm value is reduced when urea is added to the solution: Urea and formaldehyde acts as competitive denaturants as they have functional groups that forms hydrogen bond with the electronegative centers of N- bases. The presence of urea leads to competition for hydrogen bonds and sometimes favors the interaction between denaturant and the nitrogen bases instead with complementary strands resulting into separation of two strands and thereby decreasing the Tm.
5.   The Tm value is reduced when cytosine bases undergoes deamination: Since cytosine undergoes spontaneous deamination to form uracil. These “U” would end up pairing with "A" leading to decrease in GC content. Lower GC content is always directly correlated with low Tm as AT base pairs has 2 hydrogen bonds while GC base pairs have 3 hydrogen bonds.