Data Analysis Basics for Hi-Res Melting™

The power of Hi-Res Melting is augmented by analytical software that allows visual inspection of melting curve data to identify changes in the shape of the curve which indicate the presence of sequence variances in the PCR product.

Step 1. The first step in the analysis is to normalize the raw original melting data (wild type samples are shown in black, and samples with SNP alleles are shown in other colors). In some cases, samples with homozygous SNPs may be distinguished from the wild type by shifts in melting temperature. The manual analysis mode of Hi-Res Melting does not apply smoothing to the data and high fidelity data is preserved.

Step 2. The next step is to tempera­ture shift the curves at the point where all double-stranded DNA are completely denatured. Here, samples with heterozygous SNPs can be easily distinguished from the wild type by the shift in the shape of their melting curves, which often cluster into distinct groups according to genotype.

Step 3. The final step is to further analyze the shape differences by subtracting the curves from a reference curve, thus generating a Difference Plot, which helps cluster samples into groups. Alternatively, samples can be cluster automatically into groups using the Call-IT™ software (not all instruments have the Call-IT function).