Original articleSimultaneous Mutation Detection in 90 Retinal Disease Genes in Multiple Patients Using a Custom-designed 300-kb Retinal Resequencing Chip
Section snippets
Materials and Methods
For this study, we designed a custom 300-kb Affymetrix resequencing chip8(http://media.affymetrix.com/support/technical/other/customseq_design_manual.pdf, accessed April 10, 2010) capable of sequencing both forward and reverse strands of 90 retinal disease genes. For validation of the chip, we included DNA from patients with retinal diseases containing 87 known sequence changes in 25 of the genes on the chip. The study was performed in agreement with the Declaration of Helsinki. Approval of the
Custom Resequencing Chip Design
We designed a custom 300-kb Affymetrix resequencing chip, allowing for the direct sequencing of 265 000 double-stranded bases of retinal disease genes. For 90 retinal disease genes, the array covers all coding exons and 12 bases of flanking intronic sequence on either side of the exons. For 7 genes, several single nucleotide polymorphisms (SNPs) are included (Table 1). Array specifications required that 12 additional bases are added on the 5' and 3' side of each amplicon; these 24 bases are not
Discussion
This study presents a high-throughput diagnostic tool for the detection of known and new mutations in patients with inherited retinal diseases. We describe the design, validation, and use of a new 300-kb retinal resequencing chip that allows for the direct sequencing of all coding exons and flanking introns of 90 retinal disease genes. We devised a unique pooling strategy that allows for the screening of multiple patients or genes per chip, which will reduce the costs per screened individual
Acknowledgments
The authors thank Drs. M. H. Breuning, A. Plomp, L. Prick, F. Meire, and M. van Genderen for sending in DNA of patients.
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Manuscript no. 2009-1455.
Financial Disclosure(s): The author(s) have no proprietary or commercial interest in any materials discussed in this article.
Funded by the Algemene Nederlandse Vereniging ter Voorkoming van Blindheid, Landelijke Stichting voor Blinden en Slechtzienden, Rotterdamse Vereniging Blindenbelangen, Stichting Blindenpenning, Stichting Oogfonds Nederland, and Gelderse Blindenstichting.