Tetra‐primer amplification refractory mutation system (ARMS) PCR used to detect 3’UTR rs1948 mutation in CHRNB4


Anggi Laksmita Dewi(1), Dewi Kartikawati Paramita(2), Jajah Fachiroh(3*)

(1) Postgraduate student of Master of Biomedical Sciences, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Jl. Farmako Sekip, Yogyakarta 55281, Indonesia
(2) Department of Histology and Cell Biology, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Jl. Farmako Sekip, Yogyakarta 55281, Indonesia
(3) Department of Histology and Cell Biology, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Jl. Farmako Sekip, Yogyakarta 55281, Indonesia
(*) Corresponding Author


Rs1948 A>G is a single nucleotide variation (SNV) in the 3’‐UTR of CHRNB4. Genotyping the synonymous CHRNB4 rs1948 may be useful in identifying a lung cancer susceptibility gene. The study aimed to develop a simple and easy tetra‐primer amplification refractory mutation system (ARMS PCR) for CHRNB4 rs1948. The following steps were taken to optimize tetra‐primer ARMS PCR: 1) determining the gene sequence and position of a single mutation; 2) developing outer and inner primers; 3) amplification of target gene fragments via PCR using an outer primer; 4) genotyping PCR product using Sanger sequencing; 5) determining the optimal annealing temperature and PCR cycle; 6) determining optimal outer and inner primer ratio; and 7) testing the reproducibility of the PCR program and final validation with Sanger sequencing. Genotype (PCR result) was visualized with 3% agarose gel electrophoresis. Optimum condition was determined as annealing temperature of 64.8 ºC and 35 cycles, outer and inner primer ratio of 1:6, and DNA volume of 3 µL. Sanger sequencing confirmed the results of the tetra‐primer ARMS PCR and it was shown that ARMS PCR was able to identify three different variants of CHRNB4 rs1948.


CHRNB4; genetic variant; genotyping; nicotinic receptor; tobacco smoking; rs1948

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DOI: https://doi.org/10.22146/ijbiotech.64933

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