The use of spectroscopic techniques in the study of DNA stability
Accurate determination of nucleic acid thermodynamics has become increasingly important in understanding biological function as well as applications in biotechnology and pharmaceuticals. Knowledge of the thermodynamics of DNA hybridization and secondary structure formation is necessary for understanding DNA replication fidelity (1), mismatch repair efficiency (2) and the mechanism of DNA triplet repeat diseases (3). In addition, RNA folding thermodynamics are an important aspect of understanding ribozyme catalysis, as well as understanding the regulation of protein expression, mRNA stability and the mechanism of protein synthesis by the ribosome (4). With the genome sequencing era upon us (5), it will increasingly become important to predict the folding and hybridization thermodynamics of DNA and RNA, so that accurate diagnostic tests for genetic and infectious diseases can be developed. Thus, there is a need to develop a database of accurate thermodynamic parameters for different nucleic acid folding motifs (4). This chapter describes practical aspects of the application of UV absorbance temperature profiles to determine the thermodynamics of nucleic acid structural transitions. Protocols and practical advice are presented for issues not normally addressed in the primary literature but that are crucial for the determination of reliable thermodynamics, such as sequence design, sample preparation, choice of buffer, protocols for determining strand concentrations and mixing strands, design of microvolume cuvettes and cell holder, instrumental requirements, data analysis methods, and sources of error. References to the primary literature and reviews are also provided where appropriate.