Determination of Melting Temperature for Variant Detection Using dHPLC: A Comparison Between an Empirical Approach and DNA Melting Prediction Software

Abstract

Detection of DNA sequence variants by the use of denaturing high-performance liquid chromatography (dHPLC) is a relatively new method (Underhill et al., 1997) and has distinct advantages over other methods such as single-strand conformation polymorphism (SSCP), direct sequencing, and DNA chip hybridization. The dHPLC-based single-nucleotide polymorphism (SNP) screening relies on different DNA thermodynamic properties between perfectly matched base pairs in homoduplex molecules and single base-pair mismatches in heteroduplex DNAs. Separation of the two forms of duplex DNAs by dHPLC is based on ionic forces between the negatively charged DNA and the hydrophobic stationary phase, which consists of C₁₈ chains on PSDVB (polystyrene-divinylbenzene) beads coated with a positively charged ion-pairing agent (TEAA, triethylammonium acetate). Removal of the DNA from the TEAA-coated beads is dependent upon a mobile organic phase, in the form of a linear acetonitrile gradient. The major factor that influences the success of dHPLC to detect sequence variation is the thermal stability of the duplex DNA, which is determined by the melting temperature (TM₅₀), where 50% of the DNA strand is single stranded and 50% is double stranded. The TM₅₀ predicts the best probability of detecting a single base-pair change based on the altered thermodynamics it imparts to the DNA duplex. Generally, there are two ways to determine this melting temperature, either empirically or with the aid of predictive DNA melting analysis software. Such programs include the DNAMelt program located on the Stanford University DNA Sequencing and Technology Center website, MeltCalc© (Schutz and vonAhsen 1999), and WAVEMAKER®, the proprietary melting analysis software provided with the Transgenomic WAVE® dHPLC system. The goal of the current study was to determine whether currently available predictive DNA melting programs could be used to increase efficiency and throughput of SNP detection. A wide range of amplicons, differing in both size and GC composition, were selected for analysis to simulate the broad spectrum of PCR products that may be encountered during a large-scale dHPLC screening project.