Venous blood samples were obtained using K3 EDTA Vacuette tubes (Greiner Bio-One, Kremsmunster, Austria), and genomic DNA was extracted using a high salt solution according to a standard protocol [11 (link)]. Genome-wide homozygosity mapping was performed using the HumanCytoSNP-12v2.1 BeadChip (220 K; Illumina, Inc., San Diego, CA). Homozygous regions were calculated using HomozygosityMapper [12 (link)] with a lower threshold of 1 Mb. For mutation analysis specific primers were used to PCR-amplify the 26 coding exons of PROM1, including intron-exon boundaries. Primer sequences were as previously described [9 (link)]. PCR was performed in a 25 µl reaction volume in the presence of 5X Readymix (LAROVA GmbH, Teltow, Germany) and 10 pmol of each forward and reverse primers. Annealing temperature was 60 °C. Mutation screening was performed with direct sequencing with the BigDye Terminator Cycle Sequencing kit on an ABI 3130xl Genetic Analyzer (PE Applied Biosystems, Foster City, CA).
Protocol detail
Genome-wide homozygosity mapping and PROM1 mutation analysis
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Corresponding Organization :
Other organizations : Technion – Israel Institute of Technology, Rambam Health Care Campus, Tel Aviv Sourasky Medical Center, Tel Aviv University
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Variable analysis
independent variables
- Venous blood samples were obtained using K3 EDTA Vacuette tubes (Greiner Bio-One, Kremsmunster, Austria)
- Genomic DNA was extracted using a high salt solution according to a standard protocol [11]
- Genome-wide homozygosity mapping was performed using the HumanCytoSNP-12v2.1 BeadChip (220 K; Illumina, Inc., San Diego, CA)
- Homozygous regions were calculated using HomozygosityMapper [12]
- Specific primers were used to PCR-amplify the 26 coding exons of PROM1, including intron-exon boundaries
- PCR was performed in a 25 µl reaction volume in the presence of 5X Readymix (LAROVA GmbH, Teltow, Germany) and 10 pmol of each forward and reverse primers
- Annealing temperature was 60 °C
- Mutation screening was performed with direct sequencing with the BigDye Terminator Cycle Sequencing kit on an ABI 3130xl Genetic Analyzer (PE Applied Biosystems, Foster City, CA)
- Homozygous regions
- Mutations in the PROM1 gene
- Intron-exon boundaries of the PROM1 gene
- Annealing temperature of 60 °C during PCR
- No positive or negative controls were explicitly mentioned in the protocol.
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