Tetra Primer ARMS PCR Optimization to Detect Single Nucleotide Polymorphisms of the CYP2E1 Gene

  • Suhda, Saihas (Biomedical Science, Faculty of Medicine, Universitas Gadjah Mada) ;
  • Paramita, Dewi Kartikawati (Histology and Cell Biology, Faculty of Medicine, Universitas Gadjah Mada) ;
  • Fachiroh, Jajah (Histology and Cell Biology, Faculty of Medicine, Universitas Gadjah Mada)
  • Published : 2016.07.01


Single nucleotide polymorphism (SNP) detection has been used extensively for genetic association studies of diseases including cancer. For mass, yet accurate and more economic SNP detection we have optimized tetra primer amplification refractory mutation system polymerase chain reaction (ARMS PCR) to detect three SNPs in the cytochrome P450 2E1 (CYP2E1) gene locus; i.e. rs3813865, rs2070672 and rs3813867. The optimization system strategies used were (1) designing inner and outer primers; (2) determining of their optimum primer concentration ratios; and (3) determining of the optimum PCR annealing temperature. The tetra primer ARMS PCR result could be directly observed using agarose gel electrophoresis. The method succesfully determined three SNPs in CYP2E1 locus, the results being consistent with validation using DNA sequencing and restriction fragment length polymorphisms (RFLP).


  1. Khlifi R, Messaoud O, Rebai A, et al (2013). Polymorphism in the human cytochrome P450 and arylamine N-acetyltransferase: susceptibility to head and neck cancer. Bio Med Res Intl.
  2. Kwok P, Chen X (2003). Detection of single nucleotide polymorphisms. Curr Issues Mol Biol, 5, 43-60.
  3. Medrano RFV, de Oliveira CA (2014). Guidlines for the tetra primer ARMS-PCR technique development. Mol Biotechnol, 56, 599-608.
  4. Rendic S, Guengerich FP (2012). Contributions of human enzymes in carcinogen metabolism. Chem Res Toxicol, 25, 1316-83.
  5. Shastry BS (2002). SNP alleles in human disease and evolution. J Hum Gen, 47, 561-6.
  6. Xue J, Yang S, Seng S (2014). Mechanisms of cancer induction by tobacco-specific NNK and NNN. Cancers, 6, 1138-56.
  7. Ye S, Dhillon S, Ke X, et al (2001). An efficient procedure for genotyping single nucleotide polymorphisms. Nucl Acids Res, 29, 88.
  8. Zhan P, Wang J, Zhang Y, et al (2010). CYP2E1 RsaI/PstI polymorphism is associated with lung cancer risk among Asians. Lung Cancer, 69, 19-25.
  9. Akhlawat S, Sharma R, Maitra A, et al (2014). Designing, optimization, and validation of tetra primer ARMS PCR protocol for genotyping mutation in caprine Fc genes. Metagene, 2:439-49.
  10. Anantharaman D, Samant TA, Sen S, et al (2011). Polymorphisms in tobacco metabolism and DNA repair genes modulate oral precancer and cancer risk. Oral Oncol, 47, 866-72.
  11. Collins AR, Ke X (2012). Primer1: primer design web service for tetra-primer ARMS-PCR. Open Bioinformatics J, 6, 55-8.
  12. Danko IM, Chaschin NA (2005). Association of CYP2E1 gene polymorphism with predisposition to cancer development. Exp Oncol, 27, 248-56.
  13. Fareed M, Afzal M (2012). Single nucleotide polymorphism in genome-wide association of human population: a tool for broad spectrum service. Egyptian J Med Hum Gen, 14, 123-34.
  14. Feng J, Pan X, Yu J, et al (2012). Functional PstI/RsaI polymorphism in CYP2E1 is associated withthe development, progression and poor outcome of gastric cancer. PloS ONE, 7, 44478.
  15. Gray IC, Campbell DA, Spurr NK (2002). Single nucleotides polymorphisms as tools in human genetics. Hum Mol Gen, 9, 2403-8.
  16. Guo X, Zeng Y, Deng H, et al (2010). Genetic polymorphisms of CYP2E1, GSTP1, NQO1 and MPO and the risk of nasopharyngeal carcinoma in a Han Chinese population of Southern China. BMC Res Notes, 3, 1-7.
  17. Hayashi S, Watanabe J, Kawajiri K (1991). Genetic polymorphisms in the 5'-flanking region change transcriptional regulation of the human cytochrome P450IIE1 gene. J Biochem, 110, 559-65.
  18. Heathfield LJ, Dalvie S, Kalideen K, et al (2014). Novel CYP2E1 haplotype identified in a South African cohort. S Afr J Sci, 110, 9-10.
  19. Honardoost MA, Tabatabaeian H, Akbari M, et al (2014). Investigation of sensitivity, specifity and accuracy of tetra primer ARMS PCR method in comparison with conventional ARMS PCR, based on sequencing technique outcomes in IVS-II-I genotyping of beta thalassemia patients. Genes, 549, 1-6.
  20. Huang X, Chen L, Song W, et al (2012). Systemic functional characterization of cytochrome P450 2E1 promoter variants in the Chinese Han population. PLoS ONE, 7, 40883.
  21. Iversen ES, Lipton G, Clyde MA, et al. (2014). Functional annotation signatures of desease susceptibility loci improve SNP association analysis. BMC Genomics, 15, 398.
  22. Jia W, Pan Q, Qin H, et al (2009). A Case-control and a familybased association study revealing an association between CYP2E1 polymorphisms and nasopharyngeal carcinoma risk in Cantonese. Carcinogenesis, 30, 2031-6.