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

Introduction

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 absorption. 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. 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. The relationship between the Tm value and the %GC is given by the following equation, %GC = 2.44 (Tm-69.3) The Thermal Melt Analysis System for Nucleic Acids (TMSPC-8), along with the Tm Analysis software offered by Shimadzu, presents the ideal solution for determining the Tm value of a DNA solution. The versatility of this system allows for setting multiple ramp rates in a single acquisition, storing data files and routine methods for recall in future analysis, as well as providing the user with the ability to select from various algorithms for calculating the Tm values for previously stored runs. The purpose of this application news is to demonstrate the measuring capabilities of the Shimadzu TMSPC-8 temperature controlled micro cell holder for determining the Tm values of various DNA samples.

October 23, 2014 GMT