High-speed melting analysis: The effect of melting rate on small amplicon microfluidic genotyping

Robert J. Pryor, Joseph T. Myrick, Robert A. Palais, Scott O. Sundberg, Jeanette Y. Paek, Carl T. Wittwer, Ivor T. Knight

Research output: Contribution to journalArticle

9 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)1624-1632
Number of pages9
JournalClinical Chemistry
Volume63
Issue number10
DOIs
StatePublished - Oct 2017

All Science Journal Classification (ASJC) codes

  • Clinical Biochemistry
  • Biochemistry, medical

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    Pryor, R. J., Myrick, J. T., Palais, R. A., Sundberg, S. O., Paek, J. Y., Wittwer, C. T., & Knight, I. T. (2017). High-speed melting analysis: The effect of melting rate on small amplicon microfluidic genotyping. Clinical Chemistry, 63(10), 1624-1632. https://doi.org/10.1373/clinchem.2017.276147