DESAIN PRIMER UNTUK ANALISIS SEKUEN GEN MC1R, RESEPTOR GEN PENENTU PIGMENTASI RAMBUT DAN KULIT

ASTRI PUSPITA PERTIWI SETYADI, Dr. Niken Satuti Nur Handayani, M.Sc.

2021 | Skripsi | S1 BIOLOGI

Gen MC1R mengkode protein yaitu melanocortin 1 receptor yang berperan sebagai reseptor pada proses melanogenesis. Mutasi pada gen MC1R dapat meningkatan jumlah pigmen pheomelanin yang menyebabkan seseorang memiliki rambut merah, fair skin, dan freckles. Gen MC1R dapat dideteksi dengan menggunakan primer spesifik melalui Polymerase Chain Reaction (PCR) pada proses amplifikasi. Primer spesifik untuk deteksi gen MC1R tersebut dapat didesain secara in silico menggunakan software. Berdasarkan hal tersebut, penelitian ini bertujuan untuk mendesain primer spesifik secara in silico yang dapat digunakan untuk mengamplifikasi gen MC1R. Pada penelitian ini dilakukan beberapa tahap yaitu melakukan pendataan data sekuen gen MC1R dari database GenBank (accession number NC_000016.10), mendeteksi Single Nucleotide Polymorphism (SNP), mendesain primer menggunakan software Primer3Plus, menganalisis struktur sekunder (secondary structure) pada kandidat primer menggunakan program NetPrimer PREMIER Biosoft, dan selanjutnya diuji secara in vitro menggunakan metode Polymerase Chain Reaction (PCR). Single Nucleotide Polymorphism (SNP) yang dipetakan pada gen MC1R terletak pada bagian CDS dengan panjang fragmen 954 bp dan tersusun atas 317 asam amino. Bagian CDS menjadi target untuk desain primer menggunakan software Primer3Plus. Adapun protein MC1R memiliki motif asam amino yaitu asam aspartat, arginin, dan tirosin (DRY) yang berperan dalam aktivasi reseptor dan terletak pada pembatas domain transmembran 3 (TM3) dan intracellular loop 2 (IL2). Pada penelitian ini didapatkan tiga pasangan primer forward dan reverse terbaik yaitu MC1R1 (forward MC1R1_F: GAGGTGTCCATCTCTGACG dan reverse MC1R1_R: ATGAGCACCAGCATAGCC); MC1R2 (forward MC1R2_F: CATCTCTGACGGGCTCTT dan reverse MC1R2_R: ATGAGCACCAGCATAGCC); dan MC1R3 (forward MC1R3_F: ATCTCTGACGGGCTCTTCC dan reverse MC1R3_R: GCCATGAGCACCAGCATA) berdasarkan kriteria panjang primer, panjang fragmen amplikon, GC content, melting temperature (Tm), struktur sekunder primer (self-dimer, cross-dimer, dan hairpin), repeats, dan runs.

The MC1R gene encodes a protein, melanocortin 1 receptor, that acts as a receptor in the melanogenesis process. Mutations in the MC1R gene can increase in the amount of pheomelanin pigment that causes a person to have red hair, fair skin, and freckles. The MC1R gene can be detected using specific primers through Polymerase Chain Reaction (PCR) in the amplification process. Specific primers for the detection of the MC1R gene can be designed through in silico using software. Therefore, this study aimed to design specific primers through in silico that can be used to amplify the MC1R gene. In this study, several stages were conducted, firstly was to determine the MC1R gene sequence data from GenBank database (accession number NC_000016.10), secondly was to detect single nucleotide polymorphism (SNP) data, thirdly was to design specific primers using Primer3Plus software and analyze secondary structure in primer candidates using NetPrimer PREMIER Biosoft program, and then tested through in vitro using a Polymerase Chain Reaction (PCR) method. In this study, Single Nucleotide Polymorphism (SNP) mapped on the MC1R gene was located on a CDS region with a fragment length of 954 bp and consist of 317 amino acid. The CDS region was determined as the primer design target using Primer3Plus software. The MC1R protein has amino acid motifs, that is aspartic acid, arginine, and tyrosine (DRY) which play a role in receptor activation and is located at the boundary of transmembrane domain 3 (TM3) and intracellular loop 2 (IL2). The result revealed three best forward and reverse primer pairs were MC1R1 (forward MC1R1_F: GAGGTGTCCATCTCTGACG and reverse MC1R1_R: ATGAGCACCAGCATAGCC); MC1R2 (forward MC1R2_F: CATCTCTGACGGGCTCTT and reverse MC1R2_R: ATGAGCACCAGCATAGCC); and MC1R3 (forward MC1R3_F: ATCTCTGACGGGCTCTTCC and reverse MC1R3_R: GCCATGAGCACCAGCATA) based on primer length criteria, amplicon fragment length, GC content, melting temperature (Tm), primary secondary structure (selfdimer, cross-dimer, and hairpin), repeats, and runs.

Kata Kunci : Gen MC1R, desain primer, Primer3Plus, NetPrimer, PCR/ MC1R, primer design, Primer3Plus, NetPrimer, PCR