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Association of Asthma Severity and Bronchial Hyperresponsiveness With a Polymorphism: Genotyping of CTLA-4 Gene

Classification of asthma severity was based on history, symptoms, clinical features, medication need, FEV1, and PEF according to the 1997 Expert Panel Report 2. Asthma severity was classified into mild intermittent, mild persistent, moderate persistent, and severe persistent. The investigator who assessed the severity of asthma was blinded to the genotyping of the CTLA-4 gene.
DNA was extracted from peripheral blood leukocytes following standard protocols and column purified (DNA midi kit; Qiagen; Hilden, Germany). The CTLA-4 promoter polymorphism at position —318 was defined using polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) with Tru9 I restriction enzymes (Bioneer; Seoul, Korea). To amplify the target DNA in the CTLA-4 promoter, PCR was performed with oligonucleotides forward 5′-AAATGAATTGGACTGGAT-GGT-3′ and reverse 5′-TTACGAGAAAGGAAGCCGTG-3′. A 247-base pair (bp) fragment was amplified by PCR. The following conditions were applied: initial denaturation for 4 min at 94°C, then 30 cycles of 40 s at 94°C, 40 s at 60°C, and 30 s at 72°C. This was followed by a final extension for 4 min at 72°C in a thermal cycler (model 9600; PerkinElmer; Boston, MA) read more buy claritin online. PCR products were further subjected to RFLP analysis with the enzyme Tru9 I (Bioneer) and separated on a 3% agarose gel. PCR fragments with thymine at position —318 were cut into three fragments (21, 96, and 130 bp), whereas fragments with cytosine at the same position only had the restriction site at 21 bp. The CTLA-4 exon 1 position 49 (codon 17) polymorphism was defined using PCR-RFLP with the BstEII restriction enzyme (Promega; Madison, WI). PCR was carried out using forward primer 5′-AAGGCTCAGCTGAACCTGGT-3′ and reverse primer 5′-CT-GCTGAAACAAATGAAACCC-3′.