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JMD 2007, Vol. 9, No. 1
Copyright © 2007 American Society for Investigative Pathology & Association for Molecular Pathology

Technical Advance

DNA Diagnostics by Surface-Bound Melt-Curve Reactions

Linda Strömqvist Meuzelaar*, Katie Hopkins{dagger}, Ernesto Liebana{ddagger} and Anthony J. Brookes*

From the Department of Genetics, * University of Leicester, Leicester; the Salmonella Reference Unit, {dagger} Laboratory of Enteric Pathogens, Health Protection Agency Centre for Infections, London; and the Department of Food and Environmental Safety, {ddagger} Veterinary Laboratories Agency, Surrey, United Kingdom

Abstract

Melting-curve procedures track DNA denaturation in real time and so provide an effective way of assessing sequence variants. Dynamic allele-specific hybridization (DASH) is one such method, based on fluorescence, which uses heat to denature a single allele-specific probe away from one strand of a polymerase chain reaction product attached to a solid support. DASH is a proven system for research genotyping, but here we evaluate it for DNA diagnostics under two scenarios. First, for mutation scanning (resequencing), a human genomic sequence of 97 bp was interrogated with 15 probes tiled with 12-base overlaps, providing up to fourfold redundancy per base. This test sequence spanned three high-frequency single nucleotide polymorphisms, all of which were correctly revealed in 16 individuals. Second, to score multiple different mutations in parallel, 18 alterations in the gyrA gene of Salmonella were assessed in 62 strains by using wild-type- and mutation-specific probes. Both experiments were performed in a blinded manner, and all results were confirmed by sequencing. All DNA variants were unambiguously resolved in every sample, with no false-positive or false-negative signals across all of the investigations. In conclusion, DASH performs accurately and robustly when applied to DNA diagnostic challenges, including mutation scoring and mutation scanning.






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Copyright © 2007 by the American Society for Investigative Pathology and the Association for Molecular Pathology.