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J. Song, N. Smaoui, J. F. Hejtmancik, I. M. MacDonald, R. Ayyagari, S. P. Daiger, M. Brooks, A. Swaroop, X. Wang; High-Throughtput Retina-Array for Screening 93 Genes Involved in Inherited Retinal Dystrophies. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1651.
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To develop and apply a microarray-based high-throughput resequencing system to detect sequence alterations in genes related to inherited retinal dystrophies.
Affymetrix resequencing arrays were designed and manufactured to cover a total of 267,550 bases of both sense and antisense sequence spanning 93 genes containing 1,470 exons. Each gene represented on the array was associated with inherited retinal dystrophies including retinitis pigmentosa (RP), macular degeneration, cone-rod dystrophy (CRD), Usher syndrome, Stargardt disease, and Bardet-Biedl syndrome. The target DNA sequences were amplified, quantified and processed in a high-throughput fashion using an epMotion robot and LabChip 90 system. Hybridization, washing, staining and scanning were carried out using an Affymetrix GeneChip hybridization oven 645, fluidics station 450, and scanner 3000, respectively. The acquired data were first analyzed using Affymetrix GSEQ v4.1 software, and subsequently verified using an alternative software.
A total of 605 PCR assays including 355 long range (~1,500 - 6,000 bp), 235 short (<1,500 bp) range and 15 GC-rich (variable sizes) PCR assays were designed, tested and confirmed with BigDye terminator sequencing technology. Twenty-eight DNA samples including 8 control CEPH samples, and 20 patient samples with a diagnosis of RP, Usher syndrome, CRD or Stargardt disease containing 25 known sequence changes were tested in this study. The base pair call rates using the Retina-Array for the full range analysis were from 80% to 88%, and the averaged accuracy was 99.41%.
A custom designed Retina-Array for the high-content detection of mutations involved in inherited retinal dystrophies has been developed. This array will be a valuable tool for the high-throughput detection of novel mutations in a broad range of genes associated with inherited retinal disorders.
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