7.3 Stability of Secondary and Tertiary DNA Sttructures
7.3.1 Disassociation and Reassociation of double-stranded DNA
Figure 7.18: Effects of Heating and Cooling DNA
Like shown in figure 7.18, double-helical strands of DNA have the ability to reassociate and disassociate with one another.
7.3.2 Denaturation
Denaturation is the rupturing of hydrogen bonds between bases that result from increasing temperature or alteration of the H+ / OH- concentration, hence causing the two polynucleotide strands to come apart.
Increasing the pH of the solution deprotonates the nitrogen atoms of guanine and thymine.
On the contrary, decreasing the pH of the solution protonates the nitrogen atoms of adenine, guanine, and cytosine.
In short, a change in the pH of the solution can cause purine glycosidic bonds to break. For this reason, temperature is also the more practical way to denature DNA for reassociation studies.
7.3.3 Melting and melting temperature of DNA
Melting refers to the disassociation of polynucleotide strands with increasing temperature.
Figure 7.19: Melting Point of DNA
The melting temperature refers to the temperature at which half of the DNA exists as a single-strand (see figure 7.19).
Figure 7.20: Melting Point of DNA and Guanine and Cytosine Composition
The melting point of DNA is also strongly influenced by its composition of bases. In general, the more guanine and cytosine that is present in the DNA strand in question, the higher its melting temperature.
For DNA-DNA hybrids, the melting point \(T_m\) can be approximated using the Meinkoth and Wahl equation:6
\[\begin{equation} T_m = 81.5^\omicron C + 16.6\log(M) + 0.41(GC) - \frac{500}{L} \end{equation}\]
For RNA-DNA hybrids, the Casey and Davidson equation can be used instead:
\[\begin{equation} T_m = 79.8^\omicron C + 18.5\log(M) + 0.58(GC) + 11.8(GC)^2 - \frac{820}{L} \end{equation}\]
Where:
- \(M\) is the molarity of monovalent cations
- \(GC\) is the percentage of guanosine and cytosine nucleotides in DNA
- \(L\) is the length of the hybrid in base pairs
When performing PCR, the following formula for \(T_m\) can be used instead:
\[\begin{equation} T_m = 2^\omicron C(N_A + N_T) + 4^\omicron C(N_G + N_C) \end{equation}\]
Where \(N_i\) is the number of nucleotide \(i\) in the DNA solution.
7.3.4 Hyperchromic effect
Figure 7.21: Hyperchromic Effect
The hyperchromic effect refers to the stacking of base pairs as a result of melting DNA. Hence, this results in an increased absorbance at 260 nm (see figure 7.21).
This equation is only valid for DNA strands with 50 or more bases!↩︎