A High-Resolution Digital DNA Melting Platform for Robust Sequence Profiling and Enhanced Genotype Discrimination
Year: 2018
Type: article
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.
Source: SLAS TECHNOLOGY
Authors: Mridu Sinha, Hannah Mack, Todd P. Coleman, Stephanie I. Fraley
Institution: University of California San Diego
Language: English
Subfield: Biomedical Engineering
Field: Engineering
Domain: Physical Sciences
Open Access status: hybrid
Estimated APC paid: $2,070
Awards: G4582669065, G64421039
OpenAlex ID: W2797311657