A High-Resolution Digital DNA Melting Platform for Robust Sequence Profiling and Enhanced Genotype Discrimination. (December 2018)

• Record Type: Journal Article
• Authors: Sinha, Mridu; Mack, Hannah; Coleman, Todd P.; Fraley, Stephanie I.
• Is Part Of: SLAS technology. Volume 23:Number 6(2018)
• Page Start: 580
• Page End: 591
• Publication Date: 2018-12
• Subjects: point-of-care testing (POCT) -- high-throughput chemistry -- automated biology -- engineering -- informatics and software; Medical laboratory technology -- Periodicals; Laboratories -- Equipment and supplies -- Periodicals; Diagnosis, Laboratory -- Periodicals; 616.075
• DOI: 10.1177/2472630318769846
• Languages: English
• ISSNs: 2472-6303

Abstract

DNA melting analysis provides a rapid method for genotyping a target amplicon directly after PCR amplification. To transform melt genotyping into a broad-based profiling approach for heterogeneous samples, we previously proposed the integration of universal PCR and melt analysis with digital PCR. Here, we advanced this concept by developing a high-resolution digital melt platform with precise thermal control to accomplish reliable, high-throughput heat ramping of microfluidic chip digital PCR reactions. Using synthetic DNA oligos with defined melting temperatures, we characterized sources of melting variability and minimized run-to-run variations. Within-run comparisons throughout a 20, 000-reaction chip revealed that high-melting-temperature sequences were significantly less prone to melt variation. Further optimization using bacterial 16S amplicons revealed a strong dependence of the number of melting transitions on the heating rate during curve generation. These studies show that reliable high-resolution melt curve genotyping can be achieved in digital, picoliter-scale reactions and demonstrate that rate-dependent melt signatures may be useful for enhancing automated melt genotyping.