Computational protocol: Is there a relationship between endothelial nitric oxide synthase gene polymorphisms and ankylosing spondylitis?

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Protocol publication

[…] For all subjects, peripheral blood samples were collected into sterile tubes with EDTA. Genomic DNA was extracted from whole blood using standard proteinase K digestion and the salt-chloroform method. The PCR-RFLP analysis method was used to evaluate the association between three polymorphisms in the eNOS gene and AS. Three PCR primer sets were used for amplification of each polymorphic region, including the Glu298Asp (rs1799983, exon 7), −786T>C (rs2070744, promoter region) and 4b4a (intron 4, 27 bp repeat) regions. The primers were designed using the Primer3 algorithm via the primer-BLAST interface on the NCBI BLAST web site. PCR primer sequences, annealing temperatures and product sizes were as follows: Glu298Asp: (forward) 5′-GTCACGGAGACCCAGCCAATG-3′ and (reverse) 5′-GCCCTTCTTGAGAGGCTCAGGGAT-3′, 61.4 °C, 325 bp; -786T>C: (forward) 5′-AGCTAGTGGCCTTTCTCCAGCCC-3′ and (reverse) 5′-CCCAGCCCCAATTTCCTGGAACC-3′, 61.4 °C, 335 bp; and VNTR repeat region: (forward) 5′-GCCTTGGCTGGAGGAGGGGA-3′ and (reverse) 5′-TGGGGGAGAAGCAGCAGCCA-3′, 57.1 °C, 242 bp. For the Glu298Asp and −786T>C regions, PCR products were digested using MboI (Fermentas, Vilnius, Lithuania) and HpaII (New England Biolab, Hitchin, UK) restriction endonuclease enzymes, respectively. Restriction fragments were separated by electrophoresis on a gel composed of 3% agarose. For the Glu298Asp region, the T allele resulted in 195 and 130 bp bands, while the G allele resulted in single band of 225 bp. For the −786T>C region, the C allele resulted in 167, 46 and 122 bp bands, while the T allele resulted in 122 and 213 bp bands. The 27 bp VNTR repeat region in intron 4 was analyzed by direct electrophoresis on 3% agarose gel after PCR amplification. The 4b allele amplicon size was 242 bp, whereas the 4a allele amplicon size was 215 bp. HLA B27 analysis for the patient group was performed using a commercially available SSP-typing kit (Olerup; QIAGEN Vetriebs GmbH, Wien, Austria) according to the manufacturer's recommendations. SSP-typing results were visualized using 2% agarose gel electrophoresis. […]

Pipeline specifications

Software tools Primer3, Primer-BLAST, BLASTN
Application qPCR
Organisms Homo sapiens
Diseases Cardiovascular Diseases, Diabetes Mellitus, Hypertension, Liver Diseases, Spondylitis, Ankylosing
Chemicals Nitric Oxide