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DNA diagnostics by surface-bound melt-curve reactions

By Meuzelaar Stromqvist, K. Hopkins, E. Liebana and Anthony J. Brookes

Abstract

This is the final published version.\ud \ud Copyright - American Society for Investigative Pathology and the Association for Molecular Pathology. Reprinted from The Journal of Molecular Diagnostics with permission from the American Society for Investigative Pathology and Association for Molecular Pathology. Any reproduction or reuse of this material, including but not limited to reformatting, reposting, republication, translation, or other derivative works based on any portion of this article, will require separate permission from the Publisher (ASIP & AMP).\ud Definitive version of this article appears on http://jmd.amjpathol.org/Melting-curve proceedures track DNA denaturation in real time and so provide an effective was 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 single strand of a polymerase chain reaction product attached to a solid suport. 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 redundency per base. This test sequence spanned three high-frequency single nucleotide polymorphisms, all of which were corectly 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

Publisher: ASIP & AMP
Year: 2007
OAI identifier: oai:lra.le.ac.uk:2381/3733

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