PERBANDINGAN ARMS-PCR DAN ALLELE-SPECIFIC PCR DALAM OPTIMASI GENOTIPING SNP rs1998076

Abstrak

Abstrak Pendahuluan: Single nucleotide polymorphism (SNP) rs1998076 (G>A) pada kromosom 20p11 merupakan SNP yang menantang untuk digenotip karena perbedaan alel hanya satu basa dan berpotensi berada pada konteks sekuens yang meningkatkan risiko amplifikasi non-spesifik. Metode amplification-refractory mutation system polymerase chain reaction (ARMS-PCR) sering digunakan untuk genotiping SNP, namun performanya dapat sangat dipengaruhi oleh kondisi PCR, terutama suhu annealing. Penelitian ini bertujuan untuk optimasi metode terbaik untuk melihat SNP pada rs1998076 Metode: Penelitian eksperimental laboratorium membandingkan tetra-primer ARMS-PCR dan allele-specific polymerase chain reaction (AS-PCR). Validasi hasil dibandingkan dengan menggunakan Sanger sequencing Hasil: ARMS-PCR awal menunjukkan pola pita yang bervariasi, munculnya multiple bands/smear, sehingga interpretasi genotipe tidak stabil. Pada uji gradient, penurunan suhu annealing meningkatkan pita non-spesifik, sedangkan suhu lebih tinggi memperbaiki spesifisitas tetapi dapat menyebabkan melemahnya/hilangnya pita alel tertentu (indikasi allele dropout). Sebaliknya, AS-PCR menghasilkan pita tunggal yang jelas dan konsisten: sampel 02 teridentifikasi AG, sampel 03 GG, dan sampel 12 AA. Kromatogram Sanger mengonfirmasi genotipe tersebut . Kesimpulan: Tetra-primer ARMS-PCR bersifat sangat sensitif terhadap suhu annealing dan kurang reliabel untuk genotiping rs1998076 karena risiko amplifikasi non-spesifik dan allele dropout. AS-PCR dua reaksi terpisah lebih stabil dan mudah diinterpretasi sehingga lebih direkomendasikan untuk genotiping rs1998076. Kata Kunci: SNP, Genotyping, rs1998076, ARMS-PCR, AS-PCR   Abstract Background: The single nucleotide polymorphism (SNP) rs1998076 (G>A) on chromosome 20p11 is challenging to genotype because it involves a single-base substitution and may lie within a sequence context that increases the risk of nonspecific amplification. Amplification-refractory mutation system PCR (ARMS-PCR) is widely used for SNP genotyping, but its performance is highly dependent on PCR conditions, particularly annealing temperature. This study aimed to identify the most suitable PCR-based method for genotyping rs1998076. Methods: A laboratory experimental study was conducted to compare tetra-primer ARMS-PCR and allele-specific PCR (AS-PCR). Genotyping results were validated using Sanger sequencing. Results: Initial ARMS-PCR produced variable banding patterns with multiple bands/smearing, leading to unstable genotype interpretation. In annealing-temperature gradient testing, lower annealing temperatures increased nonspecific bands, whereas higher temperatures improved specificity but weakened or eliminated one allele-specific band, suggesting allele dropout. In contrast, AS-PCR generated clear and consistent single bands: sample 02 was identified as AG, sample 03 as GG, and sample 12 as AA. Sanger chromatograms confirmed these genotypes. Conclusion: Tetra-primer ARMS-PCR is highly annealing-temperature sensitive and less reliable for rs1998076 genotyping due to nonspecific amplification and potential allele dropout. Two-reaction AS-PCR is more stable and easier to interpret and is therefore recommended for genotyping rs1998076. Keywords: SNP, Genotyping, rs1998076, ARMS-PCR, AS-PCR