Establishing a cost-effective tetra-primer amplification refractory mutation system PCR for genotyping the apolipoprotein-A5 -1131T>C polymorphism: a preliminary study for genetic association analysis in the Jambi Malay population
DOI:
https://doi.org/10.22437/proca.v1i2.50262Keywords:
APOA5 -1131T>C Polymorphism, T-ARMS PCR, Genotyping, Annealing Temperature, Optimization, Cost-Effective, Jambi MalayAbstract
Background: The Apolipoprotein A5 (APOA5) gene's -1131T>C polymorphism is a significant genetic marker for dyslipidemia, with effects varying across ethnicities. Investigating this in specific populations like the Jambi Malay requires genotyping methods that are both reliable and feasible for large-scale studies, often in limited-resource settings. Methods: DNA was extracted from blood samples using a solid-phase column extraction method and subsequently diluted. Four specific primers (two outer and two inner allele-specific) for the T-ARMS PCR assay were designed. The PCR protocol was optimized by varying the annealing temperature, while other parameters like initial denaturation (95°C) and extension (72°C) were kept constant based on standard literature. Amplified products were visualized using gel electrophoresis. Results: An optimized T-ARMS PCR protocol was successfully established. The assay produced clear and distinct banding patterns on a 2.0% agarose gel, allowing for unambiguous genotype calling. Homozygous wild-type (TT) samples showed two bands (512 bp control and 355 bp T-allele), homozygous variant (CC) samples showed two bands (512 bp control and 194 bp C-allele), and heterozygous (TC) samples showed three bands (512 bp, 355 bp, and 194 bp). The assay demonstrated high specificity with no non-specific amplification at an optimal annealing temperature of 59°C. Conclusion: The developed T-ARMS PCR assay is a robust, cost-effective, and reliable method for genotyping the APOA5 rs662799 polymorphism. This tool is well-suited for large-scale molecular epidemiological studies to investigate the genetic architecture of dyslipidemia in the Jambi Malay and other resource-limited settings.
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