Qiaamp dna blood mini or midi kits

Dog owners and veterinary ophthalmologists submitted OES DNA samples to the Kennel Club Genetics Centre (previously based at the Animal Health Trust, Newmarket, UK) as buccal mucosal swabs or residual blood samples, with owner consent (AHT Ethics Approval ref: 24-2018E, University of Cambridge Ethics Approval ref: CR564). Unaffected dogs are defined as those certified clear of inherited eye disease under the BVA/KC/ISDS Eye Scheme or European/US equivalent or by a veterinary ophthalmologist (n = 48). OES MOD-affected dogs are defined as dogs diagnosed with MOD by a veterinary ophthalmologist (n = 13) or dogs diagnosed with two or more phenotypes of MOD as described in Table 1 by a veterinary ophthalmologist (n = 4). Nine OES were defined as HC-affected; OES diagnosed with cataract (not HC type) (n = 6); or OES diagnosed with an ocular abnormality not certifiable under the BVA/KC/ISDS Eye scheme (n = 6). DNA was extracted from both blood and buccal swabs using QIAamp DNA Blood Mini or Midi Kits (Qiagen, Manchester, UK).
Additional OES samples (disease status unknown) were collected and genotyped for the COL11A1 variant (n = 93) by the University of Helsinki, Finland (Ethics approval: Animal Experimental Board of Regional State Administrative Agency of Southern Finland (ESAVI/7482/04.10.07/2015, ESAVI/25696/2020).

DNA was extracted from buccal mucosal swabs using the QIAamp DNA Blood Mini or Midi Kits (Qiagen, Manchester, UK). DNA concentration and purity were determined using the NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Loughborough, UK) and/or the Qubit Fluorometer with the Qubit dsDNA broad range (BR) Assay Kit (Invitrogen, Loughborough, UK). DNA samples with concentrations < 10 nanograms per microliter (ng/μL) were concentrated using MultiScreen-PCR96 filter plates (Merck Millipore, Watford, UK) or Microcon − 30 kDa centrifugal filter units with ultracel-30 membrane (Merck Millipore, Watford, UK).