Tuberc Respir Dis > Volume 62(5); 2007 > Article
Tuberculosis and Respiratory Diseases 2007;62(5):406-416.
DOI: https://doi.org/10.4046/trd.2007.62.5.406    Published online May 1, 2007.
Comparison of PCR-RFLP and Real-Time PCR for Allelotyping of Single Nucleotide Polymorphisms of RRM1, a Lung Cancer Suppressor Gene.
Ju Yeon Jeong, Mi Ran Kim, Jun Gwang Son, Jong Pil Jung, In Jae Oh, Kyu Sik Kim, Young Chul Kim
1Medical Science Laboratory, Chonnam National University Medical School, Hwasun, Korea.
2Department of Internal Medicine, Chonnam National University Medical School, Hwasun, Korea. kyc0923@jnu.ac.kr
Abstract
BACKGROUND
Single nucleotide polymorphisms (SNPs), which consist of a substitution of a single nucleotide pair, are the most abundant form of genetic variations occurring with a frequency of approximately 1 per 1000 base pairs. SNPs by themselves do not cause disease but can predispose humans to disease, modify the extent or severity of the disease or influence the drug response and treatment efficacy. Single nucleotide polymorphisms (SNPs), particularly those within the regulatory regions of the genes often influence the expression levels and can modify the disease. Studies examining the associations between SNP and the disease outcome have provided valuable insight into the disease etiology and potential therapeutic intervention. Traditionally, the genotyping of SNPs has been carried out using polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP), which is a low throughput technique not amenable for use in large-scale SNP studies. Recently, TaqMan real-time PCR chemistry was adapted for use in allelic discrimination assays. This study validated the accuracy and utility of real-time PCR technology for SNPs genotyping METHODS: The SNPs in promoter sequence (-37 and -524) of lung cancer suppressor gene, RRM1 (ribonucleotide reductase M1 subunit) with the genomic DNA samples of 89 subjects were genotyped using both real-time PCR and PCR-RFLP. RESULTS: The discordance rates were 2.2% (2 mismatches) in -37 and 16.3% (15 mismatches) in -524. Auto-direct sequencing of all the mismatched samples(17 cases) were in accord with the genotypes read by real-time PCR. In addition, 138 genomic DNAs were genotyped using real-time PCR in a duplicate manner (two separated assays). Ninety-eight percent of the samples showed concordance between the two assays. CONCLUSION: Real-time PCR allelic discrimination assays are amenable to high-throughput genotyping and overcome many of the problematic features associated with PCR-RFLP.
Key Words: SNP, PCR-RFLP, Real-time PCR, RRM1
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