Hi-Res Melting

Hi-Res Melting is a post-PCR technique for homogeneous mutation scanning and genotyping. Through innovative engineering and imaginative development, BioFire Diagnostics has perfected Hi-Res Melting, enabling you to detect a single-base change in your sample by just melting the PCR product. Results are fast and obtained immediately after PCR by just transferring the PCR plate or tube to our instrument. With robust data acquisition, unsurpassed temperature control, and the perfect saturation dye, Hi-Res Melting is the most simple and cost-effective solution for re-sequencing and mutation detection projects.

Most re-sequencing efforts end up producing wild type (or normal) sequences. Hi-Res Melting can reduce the amount of DNA sequencing by quickly differentiating and ruling out specimens that do not have sequence variants. Further, with the use of unlabeled probe technology Hi-Res Melting can determine common or known sequence variants, finally identifying samples with unknown variants which truly need to be sequenced.

Mutations in PCR products are detected by changes in the shape of the melting curve compared to a reference sample. Below are examples of SNPs identified in two exons using the LightScanner instrument and our LCGreen Plus Dye. Superior reproducibility is demonstrated by the overlapping curves of duplicate samples for both the wild type and the mutant samples.

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).