Computational protocol: Relationship between polymorphisms in vitamin D metabolism-related genes and the risk of rickets in Han Chinese children

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Protocol publication

[…] The loci selected for this study included GC (rs4588, rs7041, rs222020, rs2282679, and rs2298849), CYP2R1 (rs10741657, rs10766197, rs12794714, rs1562902, and rs2060793), and DHCR7/NADSYN1 (rs3829251 and rs12785878), all of which have been shown to synthesize and transport vitamin D. These factors are associated with vitamin D levels.Genomic DNA was extracted from peripheral blood leukocytes using the QIAamp DNA Blood Kit (Qiagen). Twelve single-nucleotide polymorphisms (SNPs) were genotyped using the SNaPshot assay. Primer3 (http://frodo.wi.mit.edu/) was used to design primers to amplify a different-sized fragment for each SNP within a multiplex. Extension primers, again differing in length within a multiplex, were chosen from the sequence immediately up- or down-stream of each SNP. Primer interactions within the multiplex were evaluated and minimized using the AutoDimer program (http://www.cstl.nist.gov/biotech/strbase/AutoDimerHomepage/AutoDimerProgramHomepage.htm). Polymerase chain reaction (PCR) contained 10–50 ng of DNA, 1 × HotStarTaq buffer, 3 mM MgCl2, 300 μM of each dNTP, 0.08 μM of each primer, and one unit of HotStarTaq polymerase (Qiagen) in a 20-μl reaction volume. The following touchdown PCR program was used: denaturation at 95°C for 15 min, followed by 11 cycles of 94°C for 20 sec, annealing at 65°C for 40 sec (decreasing by 0.5°C per cycle), and extension at 72°C for 90 sec. This was followed by 24 cycles of denaturation at 94°C for 20 sec, annealing at 59°C for 30 sec, and extension at 72°C for 90 sec, and a final extension at 72°C for 5 min. The PCR products were purified by treatment with Exonuclease I (USB Corporation) and shrimp alkaline phosphatase (USB Corporation) at 37°C for 1 h, followed by incubation at 75°C for 15 min. The extension reaction contained 1 × ABI Prism SNaPshot Multiplex Ready Reaction Mix (Applied Biosystems), 0.5 μM of each primer, and 1 μl of each PCR product, and was carried out as recommended (Applied Biosystems). The extension PCR products were purified using 1 unit of shrimp alkaline phosphatase and then analyzed using an ABI 3130×l Genetic Analyzer. SNP calling was carried out using GeneMapperTM software v.4.0 (Applied Biosystems). For quality control, genotyping was performed without the knowledge of case/control status of the subjects, and a 5% random sample of cases and controls was genotyped twice per SNP for all SNPs by different people; the reproducibility was 100%. […]

Pipeline specifications

Software tools Primer3, AutoDimer
Databases STRBase
Application qPCR
Organisms Homo sapiens
Diseases Vitamin D Deficiency
Chemicals NAD, Vitamin D