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Jeong, Kim, Son, Jung, Oh, Kim, and Kim: Comparison of PCR-RFLP and Real-Time PCR for Allelotyping of Single Nucleotide Polymorphisms of RRM1, a Lung Cancer Suppressor Gene
Original Article
Tuberculosis and Respiratory Diseases

Tuberculosis and Respiratory Diseases 2007; 62(5): 406-416.

Published online: 31 May 2007

DOI: https://doi.org/10.4046/trd.2007.62.5.406

Comparison of PCR-RFLP and Real-Time PCR for Allelotyping of Single Nucleotide Polymorphisms of RRM1, a Lung Cancer Suppressor Gene

Ju-Yeon Jeong, M.S.1, Mi-Ran Kim, M.S.1, Jun-Gwang Son, M.D.2, Jong-Pil Jung, M.D.2, In-Jae Oh, M.D.2, Kyu-Sik Kim, M.D.2, Young-Chul Kim, M.D.2

1Medical Science Laboratory, Chonnam National University Medical School, Hwasun, Korea.

2Department of Internal Medicine, Chonnam National University Medical School, Hwasun, Korea.

Address for correspondence: Young-Chul Kim, M.D. Lung and Esophageal Cancer Clinic, Chonnam National University Medical School and Hwasun Hospital, 160 Ilsim-ri, Hwasun-gun, Jeonnam, 519-809, Korea. Phone: 82-61-379-7614, FAX: 82-61-379-7628, kyc0923@jnu.ac.kr

Received 21 February 2007       Accepted 4 May 2007

Copyright © 2007 The Korean Academy of Tuberculosis and Respiratory Diseases

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.

Keywords: SNP, PCR-RFLP, Real-time PCR, RRM1

Figures and Tables

Figure 1
Allelotyping of RRM1 by restriction enzyme digestion.
(A) RRM1-37 C/A polymorphism. Restriction enzyme Bbs I cuts when -37 base is A. Homozygous A allelle type (lane 3 from left shows complete digestion into 156 bp bands. Homozygous C allele type (lane 4 and 7) have original 217 bp bands only without digestion. The primer dimers are seen in all the lanes that are barely distinguishable from 61 bp bands in lanes with CA or AA. (B) RRM1 -524 T/C polymorphism. Restriction enzyme Apo I cuts when -524 base is T. Homozygous T allelotype (lane 2, 5 and 6 from left) shows complete digestion into 70 bp bands. Homozygous C allele type (lane 3 and 4) have original 176 bp bands only without digestion.
trd-62-406-g001
Figure 2
Allelotyping of RRM1 by real-time PCR (TaqMan probe assay) Allelic discrimination of genotyping by auto analysis software 6.0 program of Roter-Gene after RRM1 amplication. (A) genotype analysis result (B) scatter graph analysis result.
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Figure 3
Allelotyping of RRM1 gene by auto-direct sequencing.
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Table 1
Primer sequences for PCR-RFLP of -37 and -524 SNP* of RRM1† gene
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Table 2
Primer and probe sequences for real-time PCR of -37 and -524 SNP of RRM1 gene
trd-62-406-i002

*BHQ: black hole quencher.

Table 3
Comparison of allele frequency at -37 and -524 region of RRM1 gene between PCR-RFLP and RT-PCR. The results from RT-PCR were validated using Hardy-Weinberg equilibrium.
trd-62-406-i003

*Errors / Total genotypes, †Mismatched results of two samples were changed to CC→CA , CA→CC.

Table 4
Comparison of allele frequency at -37 and -524 region of RRM1 gene in duplicated RT-PCR experiments. The results from RT-PCR were validated using hardy-weinberg equilibrium.
trd-62-406-i004

*Errors / Total genotypes, †Mismatched results of two samples were CC→CA and CA→CC, ‡Mismatched results of three samples were TC→TT, TT→TC and TT→TC.

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