High-Speed Melting Analysis: The Effect of Melting Rate on Small Amplicon Microfluidic Genotyping

Abstract

Abstract BACKGROUND High-resolution DNA melting analysis of small amplicons is a simple and inexpensive technique for genotyping. Microfluidics allows precise and rapid control of temperature during melting. METHODS Using a microfluidic platform for serial PCR and melting analysis, 4 targets containing single nucleotide variants were amplified and then melted at different rates over a 250-fold range from 0.13 to 32 °C/s. Genotypes (n = 1728) were determined manually by visual inspection after background removal, normalization, and conversion to negative derivative plots. Differences between genotypes were quantified by a genotype discrimination ratio on the basis of inter- and intragenotype differences using the absolute value of the maximum vertical difference between curves as a metric. RESULTS Different homozygous curves were genotyped by melting temperature and heterozygous curves were identified by shape. Technical artifacts preventing analysis (0.3%), incorrect (0.06%), and indeterminate (0.4%) results were minimal, occurring mostly at slow melting rates (0.13–0.5 °C/s). Genotype discrimination was maximal at around 8 °C/s (2–8 °C/s for homozygotes and 8–16 °C/s for heterozygotes), and no genotyping errors were made at rates >0.5 °C/s. PCR was completed in 10–12.2 min, followed by melting curve acquisition in 4 min down to <1 s. CONCLUSIONS Microfluidics enables genotyping by melting analysis at rates up to 32 °C/s, requiring <1 s to acquire an entire melting curve. High-speed melting reduces the time for melting analysis, decreases errors, and improves genotype discrimination of small amplicons. Combined with extreme PCR, high-speed melting promises nucleic acid amplification and genotyping in < 1 min.