Microfluidic genotyping by rapid serial PCR and high-speed melting analysis

Scott O. Sundberg, Carl T. Wittwer, Renée M. Howell, Jarkko Huuskonen, Robert J. Pryor, Jared S. Farrar, Heather M. Stiles, Robert A. Palais, Ivor T. Knight

Research output: Contribution to journalArticlepeer-review

21 Scopus citations


BACKGROUND: Clinical molecular testing typically batches samples to minimize costs or uses multiplex lab-on-achip disposables to analyze a few targets. In genetics, multiple variants need to be analyzed, and different work flows that rapidly analyze multiple loci in a few targets are attractive. METHODS: We used a microfluidic platform tailored to rapid serial PCR and high-speed melting (HSM) to genotype 4 single nucleotide variants. A contiguous stream of master mix with sample DNA was pulsed with each primer pair for serial PCR and melting. Two study sites each analyzed 100 samples for F2 (c.∗97G>A), F5 (c.1601G>A), and MTHFR (c.665C>T and c.1286A>C) after blinding for genotype and genotype proportions. Internal temperature controls improved melting curve precision. The platform's liquid-handling system automated PCR and HSM. RESULTS: PCR and HSM were completed in a total of 12.5 min. Melting was performed at 0.5 °C/s. As expected, homozygous variants were separated by melting temperature, and heterozygotes were identified by curve shape. All samples were correctly genotyped by the instrument. Follow-up testing was required on 1.38% of the assays for a definitive genotype. CONCLUSIONS: Wedemonstrate genotyping accuracy on a novel microfluidic platform with rapid serial PCR and HSM. The platform targets short turnaround times for multiple genetic variants in up to 8 samples. It is also designed to allow automatic and immediate reflexive or repeat testing depending on results from the streaming DNA. Rapid serial PCR provides a flexible genetic work flow and is nicely matched to HSM analysis.

Original languageEnglish (US)
Pages (from-to)1306-1313
Number of pages8
JournalClinical chemistry
Issue number10
StatePublished - Oct 1 2014

All Science Journal Classification (ASJC) codes

  • Clinical Biochemistry
  • Biochemistry, medical


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