Thermal Analysis of DNA using the Shimadzu TMSPC-8 Temperature Controlled Accessory

The study of DNA is central to the understanding of many biological processes. DNA is a polymer made up of nucleotides containing four major bases: adenine, thymine, guanine, and cytosine. These nucleotides are held together by 3', 5'-phosphodiester bonds to form single-stranded DNA (ssDNA). The important double stranded DNA (dsDNA) is the combination of two ssDNA strands, which are held together by hydrogen bonds where adenine (A) is always bound to thymine (T) and guanine (G) is always bound to cytosine (C). The AT bond is comprised of two hydrogen bonds where the GC pair is bound by three hydrogen bonds and is, therefore, a stronger bond.

DNA exhibits absorption properties in the UV range at 260 nm because of the presence of these aromatic bases. This feature provides useful information into the base-pair composition of DNA because structural changes such as helix unwinding affect the extent of absorption1.

If dsDNA samples are treated with denaturing agents such as heat, alkali, or organic solvents, the H-bonds can be broken and the absorbance values at 260 nm will change. For example, Figure 1 shows a typical thermal denaturation curve for DNA, which shows changes in absorbance as the temperature of the sample is increased. The total increase in absorption for this type of analysis is usually on the order of 40% and occurs over a small temperature range. The temperature corresponding to the midpoint of the absorption increase is defined as Tm, the transition, or melting temperature. The Tm is the temperature where 50% of the base-pair H-bonds in the dsDNA no longer hold the two strands together.