title: Microfluidic genotyping by rapid serial PCR and high-speed melting analysis.
abstract: BACKGROUND
  Clinical molecular testing typically batches samples to minimize costs or uses multiplex lab-on-a-chip 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
  We demonstrate 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.
authors.affiliation: Department of Pathology, University of Utah Health Sciences Center, Salt Lake City, UT; current address: Canon U.S. Life Sciences, Newport News, VA;
authors.forename: Scott O
authors.initials: SO
authors.lastname: Sundberg
authors.affiliation: Department of Pathology, University of Utah Health Sciences Center, Salt Lake City, UT; carl.wittwer@path.utah.edu.
authors.forename: Carl T
authors.initials: CT
authors.lastname: Wittwer
authors.affiliation: Canon U.S. Life Sciences, Rockville, MD;
authors.forename: Renée M
authors.initials: RM
authors.lastname: Howell
authors.affiliation: Canon U.S. Life Sciences, Rockville, MD;
authors.forename: Jarkko
authors.initials: J
authors.lastname: Huuskonen
authors.affiliation: Department of Pathology, University of Utah Health Sciences Center, Salt Lake City, UT;
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Pryor
authors.affiliation: Department of Pathology, University of Utah Health Sciences Center, Salt Lake City, UT;
authors.forename: Jared S
authors.initials: JS
authors.lastname: Farrar
authors.affiliation: Canon U.S. Life Sciences, Rockville, MD;
authors.forename: Heather M
authors.initials: HM
authors.lastname: Stiles
authors.affiliation: Department of Mathematics, Utah Valley University, Orem, UT.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Palais
authors.affiliation: Canon U.S. Life Sciences, Rockville, MD;
authors.forename: Ivor T
authors.initials: IT
authors.lastname: Knight
mesh_terms: D004247:DNA / Q000235:genetics; D004867:Equipment Design; D005165:Factor V / Q000235:genetics; D005838:Genotype; D060005:Genotyping Techniques / Q000295:instrumentation / Q000379:methods*; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D042965:Methylenetetrahydrofolate Reductase (NADPH2) / Q000235:genetics; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D044366:Transition Temperature
chemical_list: D005165:Factor V; D004247:DNA; D042965:Methylenetetrahydrofolate Reductase (NADPH2)