Oragene dna saliva collection kit

Genomic DNA was extracted from either blood or saliva, then sent as coded samples to U.C. Davis for evaluation. Saliva samples were collected using Oragene DNA saliva collection kit (DNA Genotek, Ontario, Canada) according to the manufacturer's protocol. Genomic DNA was extracted from peripheral blood leukocytes using the Qiagen QIAamp DNA Blood Reagent Kit according to the manufacturer's protocol. For PCR analysis, genomic DNA concentrations were quantified by spectrophotometry at 260 nm using a Nanodrop spectrometer then diluted to 10 ng/µl in 50.0 µM Tris buffer (pH 8.0) containing yeast RNA (20 µg/ml, functioning as a nucleic acid carrier).

Genomic DNA was extracted using standard methods from whole blood and saliva samples. Briefly, FlexiGene DNA Kit (Qiagen, Germantown, MD, USA, Catalogue number 51206) was used for genomic DNA extraction from buffy coats isolated from whole blood samples according to the manufacturer’s protocol; prepIT-L2P DNA extraction kit (DNA Genotek, Kanata, ON, Canada, Catalogue number PT-L2P-45) was used for genomic DNA extraction from saliva samples collected with Oragene^® DNA saliva collection kit (DNA Genotek, Canada, Catalogue number OG-500) according to the manufacturer’s protocol. The manufacturer’s protocol for genomic DNA extraction is described in further detail in Supplementary Materials and Methods S1. The Illumina Infinium OncoArray 500 K BeadChip was used for genotyping DNA samples [24 (link)]. Detailed information on genotype calling and quality control has been described previously [20 (link)]. Imputation was performed in two parts: SHAPEIT (v2.r904) was used for phasing [25 (link),26 (link)] and IMPUTE2 (v2.3.2) was used for the imputation [27 (link)]. The October 2014 (version 3) release of the 1000 Genomes Project was the reference panel used [28 (link)]. All chromosomal positions described are in reference to the human genome assembly GRCh37 (hg19).

The study adhered to the tenets of the Declaration of Helsinki and was approved by the Southern Adelaide Clinical Human Research Ethics Committee, Adelaide, Australia, and the Royal Victorian Eye and Ear Hospital (RVEEH) Human Research and Ethics Committee, Melbourne, Australia. The probands in each family were recruited from the eye clinic at Flinders Medical Centre (Adelaide), the Women’s and Children’s Hospital (Adelaide), the Royal Children’s Hospital (Melbourne), or the Royal Victorian Eye and Ear Hospital (Melbourne). Written informed consent was obtained from all participants or their guardians if they were under 18 years old. A detailed family history was obtained and additional affected and unaffected family members were invited to participate in the study. An ophthalmologist examined all available family members.
Genomic DNA was extracted from either peripheral whole blood using a QiaAmp DNA Blood Maxi Kit (Qiagen, Hilden, Germany) or from saliva using an Oragene DNA saliva collection kit (DNA Genotek, Ontario, Canada) according to the manufacturers’ protocols. Participants were included in this study if they reported a family history of pediatric cataract and if a causative mutation had not previously been detected in the family by other means.