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Helen L. Griffiths, Christina Lim, Charles Pierce, Sam Khandhadia, Geeta Menon, IV, Heather Thomson, Angela Cree, Andrew Lotery; Development of in-house High Resolution Melt Assays for Age-related Macular Degeneration. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3316. doi: https://doi.org/.
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To screen donor eye tissue and individual patients affected with Age Related Macular Degeneration (AMD) for multiple known mutations using an in-house High Resolution Melt Curve (HRM) analysis platform. This will allow phenotype-genotype correlations to be made for basic research studies and enable development of individualised medicine and lifestyle modification for affected individuals.
10 assays were designed for High Resolution Melt Curve analysis for use on a Corbett Rotor-Gene 6000. Primers were supplied by Primer Design Ltd. qPCR reactions were run using SYTO9 fluorescent dye and a SensiMix SYBR No-ROX One-Step Kit from Bioline. Gene name and SNP reference sequence number are detailed in the table below. SNP assays were validated using DNA samples previously genotyped and sequenced for known AMD polymorphisms/ mutations.
HRM analysis displayed a shift in the melting temperature of polymorphic DNA samples allowing accurate visual discrimination between wild-type, homozygous & heterozygous alleles, as shown for the SERPING1 SNP rs2511989 in the graph below.
This could be a reliable and cost effective technique for screening multiple AMD associated polymorphisms in small cohorts in a laboratory setting.
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