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Tetra Primer ARMS PCR Optimization to Detect Single Nucleotide Polymorphism of the KLF14 Gene

DOI: 10.4236/oalib.1104145, PP. 1-14

Subject Areas: Diabetes & Endocrinology

Keywords: rs972283 (A/G), KLF14 Gene, Type 2 Diabetes Mellitus

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GWAS (genome-wide association studies) have associated Type 2 Diabetes Mellitus with the single nucleotide polymorphism (SNPs) rs972283 (A/G) of KLF14 gene with in a global population. The most common techniques used to analyze SNPs are time consuming, multi-step process and require expensive instruments. Therefore, in order to overcome these problems, we have developed a new, rapid and cost effective T-ARMS PCR assay to genotype rs972283. However, the optimization step can be hardworking and laborious. Hence, we propose to demonstrate and discuss critical steps for its development, in a way to provide useful information. In a first step, we design and validate two specific primer pair for T-ARMS PCR. Later, the amplification conditions were optimized for DNA concentration, annealing temperature, Taq DNA polymerase units and primers concentration. The last one was considered the main interference factor for a correct amplification and appropriate band intensity. Finally, the results obtained by T-ARMS PCR were concordant with sequencing. T-ARMS PCR assay developed in our laboratory for genotyping rs972283 (A/G) of KLF14 gene is time saving and cost-effective compared to the available methods used for SNP studies.

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Zabala, A. S. , Gomez, M. E. V. , Alvarez, M. F. and Siewert, S. (2017). Tetra Primer ARMS PCR Optimization to Detect Single Nucleotide Polymorphism of the KLF14 Gene. Open Access Library Journal, 4, e4145. doi:


[1]  Fareed, M. and Afzal, M. (2012) Single Nucleotide Polymorphism in Genome-Wide Asso-ciation of Human Population: A Tool for Broad Spectrum Service. Egyptian Journal of Medical Human Genetics, 14, 123-134.
[2]  Iversen, E.S., Lipton, G., Clyde, M.A. and Monteiro, A.N. (2014) Functional Annotation Signatures of Disease Susceptibility Loci Improve SNP Association Analysis. BMC Genomics, 15, 398.
[3]  de Assuncao, T.M., Lomberk, G., Cao, S., Yaqoob, U., Mathison, A., Simonetto, D.A., et al. (2014) New Role for Kruppel-Like Factor 14 as a Transcriptional Activator Involved in the Generation of Signaling Lipids. The Journal of Biological Chemistry, 289, 15798-15809.
[4]  Voight, B.F., Scott, L.J., Steinthors-dottir, V., Morris, A.P., Dina, C., Welch, R.P., et al. (2010) Twelve Type 2 Diabetes Sus-ceptibility Loci Identified through Large-Scale Association Analysis. Nature Genetics, 42, 579-589.
[5]  Ohshige, T., Iwata, M., Omori, S., Tanaka, Y., Hirose, H., Kaku, K., Maegawa, H., Watada, H., Kashiwagi, A., Kawamori, R., Tobe, K., Kadowaki, T., Nakamura, Y. and Maeda, S. (2011) Association of New Loci Identified in European Genome-Wide Association Studies with Susceptibility to Type 2 Diabetes in the Japanese. PLoS One, 6, e26911.
[6]  Small, K.S., Hedman, A.K., Grundberg, E., Nica, A.C., Thorleifsson, G., Kong, A., et al. (2011) Identification of an Imprinted Master Trans Regulator at the KLF14 Locus Related to Multiple Metabolic Phe-notypes. Nature Genetics, 43, 561-564.
[7]  Wang, J., Zhang, J., Shen, J., Hu, D., Yan, G., Liu, X., Xu, X., Pei, L., Li, Y. and Sun, C. (2014) As-sociation of KCNQ1 and KLF14 Polymorphisms and Risk of Type 2 Diabetes Mellitus: A Global Meta-Analysis. Human Immunology, 75, 342-347.
[8]  Gao, K., Wang, J., Li, L., Zhai, Y., Ren, Y., You, H., Wang, B., Wu, X., Li, J., Liu, Z., Li, X., Huang, Y., Luo, X.P., Hu, D., Ohno, K. and Wang, C. (2016) Polymorphisms in Four Genes (KCNQ1 rs151290, KLF14 rs972283, GCKR rs780094 and MTNR1B rs10830963) and Their Correlation with Type 2 Diabetes Mellitus in Han Chinese in Henan Province, China. International Journal of Environmental Research and Public Health, 26.
[9]  Kwok, P.Y. and Chen, X. (2003) Detection of Single Nucleotide Polymorphisms. Current Issues in Molecular Biology, 5, 43-60.
[10]  Medrano, R.F.V. and de Oliveira, C.A. (2014) Guidlines for the Tetra Primer ARMS-PCR Technique Development. Molecular Biotechnology, 56, 599-608.
[11]  Newton, C.R., Graham, A., Heptinstall, L.E., Powell, S.J., Summers, C. and Kalsheker, N. (1989) Analysis of Any Point Mutation in DNA: The Amplification Refractory Mutation System (ARMS). Nucleic Acids Research, 17, 2503-2516.
[12]  Okayama, N., Fujimura, K., Nakamura, J., Suehiro, Y., Hamanaka, Y. and Hinoda, Y. (2004) Evaluation of a New Efficient Procedure for Single-Nucleotide Polymorphism Genotyping: Tetra-Primer Amplification Refractory Mutation System-Polymerase Chain Reaction. Clinical Chemistry and Laboratory Medi-cine, 42, 13-16.
[13]  Ye, S., Dhillon, S., Ke, X., Collins, A.R. and Day, I.N. (2001) An Efficient Procedure for Genotyping Single Nu-cleotide Polymorphisms. Nucleic Acids Research, 29, E88-8.
[14]  Alyethodi, R.R., Singh, U., Kumar, S., Deb, R., Alex, R., Sharma, S., Sengar, G.S. and Prakash, B. (2016) Development of a Fast and Economical Genotyping Protocol for Bovine Leukocyte Adhesion Deficiency (BLAD) in Cattle. Springerplus, 5, 1442.
[15]  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, 439-449.
[16]  Ye, S., Humphries, S. and Green, F. (1992) Allele Specific Amplification by Tetra-Primer PCR. Nucleic Acids Research, 20, 1152.
[17]  Little, S. (1997) ARMS Analysis of Point Mutations. In: Taylor, G.R., Ed., Laboratory Methods for the Detection of Mutations and Polymorphisms in DNA, CRC Press, Boca Raton, 45-51.
[18]  Breslauer, K.J., Frank, R., Blocker, H. and Marky, L.A. (1986) Predicting DNA Duplex Stability from the Base Sequence. Proceedings of the National Academy of Sciences of the USA, 83, 3746-3750.
[19]  Rychlik, W., Spencer, W.J. and Rhoads, R.E. (1990) Optimization of the Annealing Temperature for DNA Amplification in Vitro. Nucleic Acids Research, 18, 6409-6412.
[20]  Rozen, S. and Skaletsky, H. (2000) Primer3 on the WWW for General Users and for Biologist Programmers. Methods in Molecular Biology, 132, 365-386.
[21]  Chiapparino, E., Lee, D. and Donini, P. (2004) Genotyping Single Nucleotide Polymorphisms in Barley Tetra-Primer ARMS-PCR. Genome, 47, 414-420.
[22]  Hube, F., Reverdiau, P., Iochmann, S. and Gruel, Y. (2005) Improved PCR Method for Amplification of GC-Rich DNA Sequenc-es. Molecular Biotechnology, 31, 81-84.
[23]  McDowell, D.C., Burns, N.A. and Parkes, H.C. (1998) Localized Sequence Regions Possessing High Melting Temperatures Prevent the Amplification of a DNA Mimic in Competitive PCR. Nucleic Acids Resarch, 26, 3340-3347.
[24]  Garce’s-Claver, A., Fellman, S.M., Gil-Ortega, R., Jahn, M. and Arnedo-Andre’s, M.S. (2007) Identification, Validation and Survey of a Single Nucleotide Polymorphism (SNP) Associated with Pungency in Capsicum spp. TAG Theoretical and Applied Genetics, 115, 907-916.
[25]  Andrew, C. and Xiayi, K. (2012) Primer1: Primer Design Web Service for Tetra-Primer ARMS-PCR. The Open Bioinformatics Journal, 6, 55-58.


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