DESAIN PRIMER GEN EDNRB EKSON 4 DAN OPTIMASI PCR UNTUK ANALISIS MUTASI GEN PADA HIRSCHSPRUNG DISEASE
DOI:
https://doi.org/10.30605/biogenerasi.v10i2.5954Keywords:
EDNRB gene, primer design, PCR optimization, Hirschsprung Disease (HD)Abstract
EDNRB gene is a gene located on chromosome 13q22, plays a role in the signaling pathway during the development of the ENS intestinal nerves. Mutations in the EDNRB gene in humans are associated with Hirschsprung Disease (HD). Mutations in the EDNRB gene are the second most common cause after the RET gene in cases of HD with a percentage of 5–10%. To detect mutations, PCR products are needed. Primers are one of the basic components of PCR, while optimization of annealing temperature and optimization of primer concentration are factors in the success of primer attachment to the target gene. This study aims to design EDNRB exon 4 gene primers and determine the optimum annealing temperature and primer concentration to amplify the EDNRB gene. Primer design with Geneious Prime, and good primer criteria are analyzed in silico. Optimization of annealing temperature and primer concentration is carried out with gradient PCR and variations in primer concentration. The results of this study obtained the best primers, namely the forward primer 5'- CAGTAAGTGTGGCCTGAAAG-3' and the reverse primer 5'-GTGGAACCGAAGTGACTAGA-3', which amplified the EDNRB gene exon 4. The optimum PCR conditions for amplification of exon 4 of the EDNRB gene were at an annealing temperature of 57.2 ℃ and at a primer concentration of 0.5 μM.
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References
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Auricchio, A., Griseri, P., Carpentieri, M. L., Betsos, N., Staiano, A., Tozzi, A., ... & Ceccherini, I. (1999). Double Heterozygosity for A RET Substitution Interfering with Splicing and An EDNRB Missense Mutation in Hirschsprung Disease. The American Journal of Human Genetics, 64(4), 1216-1221.
Badriyya, E., & Achyar, A. (2021). Primer Design of SNP rs4506565 Transcription Factor 7 like 2 (TCF7L2) Gene to Detect Type-2 Diabetes Mellitus. In 2nd International Conference on Contemporary Science and Clinical Pharmacy 2021 (ICCSCP 2021) (pp. 197-202). Atlantis Press.
Chuang, L. Y., Cheng, Y. H., & Yang, C. H. (2013). Specific primer design for the polymerase chain reaction. Biotechnology letters, 35, 1541-1549.
Diposarosa, R., Bustam, N. A., Sahiratmadja, E., Susanto, P. S., & Sribudiani, Y. (2021). Literature Review: Enteric Nervous System Development, Genetic and Epigenetic Regulation in The Etiology of Hirschsprung's Disease. Heliyon, 7(6).
Grada, A., & Weinbrecht, K. (2013). Next-generation sequencing: methodology and application. Journal of Investigative Dermatology, 133(8), 1-4.
Green, M. R., & Sambrook, J. (2019). Polymerase Chain Reaction. Cold Spring Harbor Protocols, 2019(6), Pdb-Top095109.
Gunadi, Kalim, A. S., Marcellus, Budi, N. Y. P., & Iskandar, K. (2022). The impact of NRG1 expressions and methylation on multifactorial Hirschsprung disease. BMC pediatrics, 22(1), 216.
Herman, H., Natalya, L. N., Berampu, S. M., & Roslim, D. I. (2017). Optimasi Suhu Annealing untuk Primer G-Ssr dan Est-Ssr pada Kacang Hijau (Vigna radiata L.). Dinamika Pertanian, 33(1), 95-102.
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Luzón‐Toro, B., Villalba‐Benito, L., Torroglosa, A., Fernández, R. M., Antiñolo, G., & Borrego, S. (2020). What Is New About the Genetic Background of Hirschsprung Disease? Clinical Genetics, 97(1), 114-124.
Mahdieh, N., & Rabbani, B. (2013). An Overview of Mutation Detection Methods in Genetic Disorders. Iranian Journal of Pediatrics, 23(4), 375.
Majdawati, A. (2009). Peran Pemeriksaan Barium Enema Pada Penderita Megacolon Congenital (Hirschprung Diseases). Mutiara Medika: Jurnal Kedokteran Dan Kesehatan, 9(2), 64-72
Mardiana, A., Dhanti, K. R., Kurniawan, K., & Sulistyowati, R. (2023). Optimasi Konsentrasi Primer Dan Suhu Annealing Dalam Mendeteksi Gen Blaz Pada Bakteri Methicillin Resistant Staphylococcus Aureus Di Udara: Optimization of Primary Concentration and Annealing Temperature in Detecting Blaz Gene in Airborne Methicillin-resistant Staphylococcus aureus Bacteria. Borneo Journal of Medical Laboratory Technology, 6(1), 389-393.
Masnaini, M., Achyar, A., Chatri, M., Putri, D. H., & Ahda, Y. (2023). Primer Design and Optimization of PCR Methods for Detecting Mixed Rat Meat in Food. In Proceedings of the 3rd International Conference on Biology, Science and Education (IcoBioSE 2021) (Vol. 32, p. 282). Springer Nature.
Medline plus, 2022. EDNRB Gene. United States government. https://medlineplus.gov/genetics/gene/ednrb/#conditions (di Akses tanggal 12 November 2024).
Ramadhanil, S., Putri, F. R., & Farma, S. A. (2023). Desain primer dan analisis in silico gen glutathione peroxidase-1 pada Rattus norvegicus. Tarumanagara Medical Journal, 5(2), 374-383.
Sanchez-Mejias, A., Fernández, R. M., Lopez-Alonso, M., Antiñolo, G., & Borrego, S. (2010). New roles of EDNRB and EDN3 in the pathogenesis of Hirschsprung disease. Genetics in Medicine, 12(1), 39-43.
Saraswati, H., Seprianto, S., & Wahyuni, F. D. (2019). Desain Primer Secara in Silico Untuk Amplifikasi Gen Cryiii Dari Bacillus Thuringiensis Isolat Lokal. Indonesian Journal of Biotechnology and Biodiversity, 3(1), 33-38.
Sasmito, D. E. K., Kurniawan, R., & Muhimmah, I. (2014). Karakteristik Primer Pada Polymerase Chain Reaction (PCR) Untuk Sekuensing DNA: Mini Review. In Seminar Nasional Informatika Medis (Snimed) (Pp. 93-102).
Septiasari, N. P. S., Dwijastuti, N. M. S., Handayani, N. S., & Darmayanti, P. D. (2025). Desain primer secara in silico dan optimasi PCR untuk deteksi gen penyandi ovary ecdysteroidogenic hormone (OEH) pada nyamuk Aedes aegypti yang terinfeksi Wolbachia: In silico primer design and PCR optimization for detection of the gene encoding ovary ecdysteroidogenic hormone (OEH) in Aedes aegypti mosquitoes infected with Wolbachia. Jurnal Entomologi Indonesia, 22(1), 9-16.
Sergi, C. M., Caluseriu, O., McColl, H., & Eisenstat, D. D. (2017). Hirschsprung’s disease: clinical dysmorphology, genes, micro-RNAs, and future perspectives. Pediatric research, 81(1), 177-191.
Setiadi, Q. H., & Haikal, Z. (2021). Total Colonic Aganglionosis: Dilema Diagnosis dan Dampak Jangka Panjang. Jurnal Kedokteran, 10(3), 531-536.
Setiani, N. A., Tritama, E., & Tresnawulansari, A. (2021). Optimasi Optical Density (Od) Pada Isolasi Genom Salmonella Typhi Menggunakan Genomic Dna Purification Kit. Jurnal Sains Dan Teknologi Farmasi Indonesia, 10(1), 35-43.
Setyawati, R., & Zubaidah, S. (2021). Optimasi konsentrasi primer dan suhu annealing dalam mendeteksi gen leptin pada sapi peranakan ongole (PO) menggunakan polymerase chain reaction (PCR). Indonesian Journal of Laboratory, 4(1), 36-40.
Silambi, A., Setyawati, T., & Langitan, A. (2020). Case report: Hirschsprung disease. Jurnal Medical Profession (MedPro), 2(1), 36-40.
Stujanna, E. N., Ningsih, S. S., Avissa, R., Ayu, N. P., Nurussofa, Z., Djuarna, D. J., ... & Sukarya, W. S. (2022). Collagen-VI Specific Primer Design Identification in Rats (Rattus norvegicus) Pancreas. Indonesian Journal of Medical Laboratory Science and Technology, 4(1), 60-70.
Tjan, A. (2021). Radiology perspective one-year study of Hirschsprung Disease. Folia Medica Indonesiana, 57(1), 41-45.
Violita, V., Achyar, A., Zulyusri, Z., Atifah, Y., Putri, D. H., & Nabilah, R. (2024). Primer Design and Optimization of Annealing Temperature for Gene Amplification GSTL2 on Rice. Al-Kauniyah: Jurnal Biologi, 17(2), 377-386.
Wei, F., Ge, Y., Li, W., Wang, X., & Chen, B. (2020). Role of endothelin receptor type B (EDNRB) in lung adenocarcinoma. Thoracic cancer, 11(7), 1885-1890.
Widowati, T., Melhem, S., Patria, S. Y., De Graaf, B. M., Sinke, R. J., Viel, M., ... & Sribudiani, Y. (2016). RET And EDNRB Mutation Screening in Patients with Hirschsprung Disease: Functional Studies and Its Implications for Genetic Counseling. European Journal of Human Genetics, 24(6), 823-829
Wu, T. T., Tsai, T. W., Chu, C. T., Lee, Z. F., Hung, C. M., Su, C. C., ... & Li, C. (2005). Low RET Mutation Frequency and Polymorphism Analysis of The RET And EDNRB Genes in Patients with Hirschsprung Disease In Taiwan. Journal Of Human Genetics, 50(4), 168-174.
Zheng, Y., Lan, C., Wang, N., Xu, X., Hu, T., Wu, Q., ... & Li, C. (2020). Significant Association of rs2147555 Genetic Polymorphism in the EDNRB Gene with Hirschsprung Disease in Southern Chinese Children. BioMed Research International, 2020(1), 5956412.
Ahda, Y., Pranata, A., & Achyar, A. (2021). The Characteristic of Luciferase cDNA of Lamprigera sp. (Lampyridae: Coleoptera). In Journal of Physics: Conference Series (Vol. 1940, No. 1, p. 012074). IOP Publishing.
Aulia, N., Ahda, Y., Achyar, A., & Putri, D. H. (2023). Desain Primer dan Optimasi Suhu Annealing untuk Amplifikasi Gen RET. BIOSEL (Biology Science and Education): Jurnal Penelitian Science dan Pendidikan, 12(1), 70-77.
Auricchio, A., Griseri, P., Carpentieri, M. L., Betsos, N., Staiano, A., Tozzi, A., ... & Ceccherini, I. (1999). Double Heterozygosity for A RET Substitution Interfering with Splicing and An EDNRB Missense Mutation in Hirschsprung Disease. The American Journal of Human Genetics, 64(4), 1216-1221.
Badriyya, E., & Achyar, A. (2021). Primer Design of SNP rs4506565 Transcription Factor 7 like 2 (TCF7L2) Gene to Detect Type-2 Diabetes Mellitus. In 2nd International Conference on Contemporary Science and Clinical Pharmacy 2021 (ICCSCP 2021) (pp. 197-202). Atlantis Press.
Chuang, L. Y., Cheng, Y. H., & Yang, C. H. (2013). Specific primer design for the polymerase chain reaction. Biotechnology letters, 35, 1541-1549.
Diposarosa, R., Bustam, N. A., Sahiratmadja, E., Susanto, P. S., & Sribudiani, Y. (2021). Literature Review: Enteric Nervous System Development, Genetic and Epigenetic Regulation in The Etiology of Hirschsprung's Disease. Heliyon, 7(6).
Grada, A., & Weinbrecht, K. (2013). Next-generation sequencing: methodology and application. Journal of Investigative Dermatology, 133(8), 1-4.
Green, M. R., & Sambrook, J. (2019). Polymerase Chain Reaction. Cold Spring Harbor Protocols, 2019(6), Pdb-Top095109.
Gunadi, Kalim, A. S., Marcellus, Budi, N. Y. P., & Iskandar, K. (2022). The impact of NRG1 expressions and methylation on multifactorial Hirschsprung disease. BMC pediatrics, 22(1), 216.
Herman, H., Natalya, L. N., Berampu, S. M., & Roslim, D. I. (2017). Optimasi Suhu Annealing untuk Primer G-Ssr dan Est-Ssr pada Kacang Hijau (Vigna radiata L.). Dinamika Pertanian, 33(1), 95-102.
Kemenkes, 2017. Pedoman Nasional Pelayanan Kedokteran Tata Laksana Penyakit Hirschprung. Kementrian Kesehatan Republik Indonesia.
Lorenz, T. C. (2012). Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies. JoVE (Journal of Visualized Experiments), (63), e3998.
Luzón‐Toro, B., Villalba‐Benito, L., Torroglosa, A., Fernández, R. M., Antiñolo, G., & Borrego, S. (2020). What Is New About the Genetic Background of Hirschsprung Disease? Clinical Genetics, 97(1), 114-124.
Mahdieh, N., & Rabbani, B. (2013). An Overview of Mutation Detection Methods in Genetic Disorders. Iranian Journal of Pediatrics, 23(4), 375.
Majdawati, A. (2009). Peran Pemeriksaan Barium Enema Pada Penderita Megacolon Congenital (Hirschprung Diseases). Mutiara Medika: Jurnal Kedokteran Dan Kesehatan, 9(2), 64-72
Mardiana, A., Dhanti, K. R., Kurniawan, K., & Sulistyowati, R. (2023). Optimasi Konsentrasi Primer Dan Suhu Annealing Dalam Mendeteksi Gen Blaz Pada Bakteri Methicillin Resistant Staphylococcus Aureus Di Udara: Optimization of Primary Concentration and Annealing Temperature in Detecting Blaz Gene in Airborne Methicillin-resistant Staphylococcus aureus Bacteria. Borneo Journal of Medical Laboratory Technology, 6(1), 389-393.
Masnaini, M., Achyar, A., Chatri, M., Putri, D. H., & Ahda, Y. (2023). Primer Design and Optimization of PCR Methods for Detecting Mixed Rat Meat in Food. In Proceedings of the 3rd International Conference on Biology, Science and Education (IcoBioSE 2021) (Vol. 32, p. 282). Springer Nature.
Medline plus, 2022. EDNRB Gene. United States government. https://medlineplus.gov/genetics/gene/ednrb/#conditions (di Akses tanggal 12 November 2024).
Ramadhanil, S., Putri, F. R., & Farma, S. A. (2023). Desain primer dan analisis in silico gen glutathione peroxidase-1 pada Rattus norvegicus. Tarumanagara Medical Journal, 5(2), 374-383.
Sanchez-Mejias, A., Fernández, R. M., Lopez-Alonso, M., Antiñolo, G., & Borrego, S. (2010). New roles of EDNRB and EDN3 in the pathogenesis of Hirschsprung disease. Genetics in Medicine, 12(1), 39-43.
Saraswati, H., Seprianto, S., & Wahyuni, F. D. (2019). Desain Primer Secara in Silico Untuk Amplifikasi Gen Cryiii Dari Bacillus Thuringiensis Isolat Lokal. Indonesian Journal of Biotechnology and Biodiversity, 3(1), 33-38.
Sasmito, D. E. K., Kurniawan, R., & Muhimmah, I. (2014). Karakteristik Primer Pada Polymerase Chain Reaction (PCR) Untuk Sekuensing DNA: Mini Review. In Seminar Nasional Informatika Medis (Snimed) (Pp. 93-102).
Septiasari, N. P. S., Dwijastuti, N. M. S., Handayani, N. S., & Darmayanti, P. D. (2025). Desain primer secara in silico dan optimasi PCR untuk deteksi gen penyandi ovary ecdysteroidogenic hormone (OEH) pada nyamuk Aedes aegypti yang terinfeksi Wolbachia: In silico primer design and PCR optimization for detection of the gene encoding ovary ecdysteroidogenic hormone (OEH) in Aedes aegypti mosquitoes infected with Wolbachia. Jurnal Entomologi Indonesia, 22(1), 9-16.
Sergi, C. M., Caluseriu, O., McColl, H., & Eisenstat, D. D. (2017). Hirschsprung’s disease: clinical dysmorphology, genes, micro-RNAs, and future perspectives. Pediatric research, 81(1), 177-191.
Setiadi, Q. H., & Haikal, Z. (2021). Total Colonic Aganglionosis: Dilema Diagnosis dan Dampak Jangka Panjang. Jurnal Kedokteran, 10(3), 531-536.
Setiani, N. A., Tritama, E., & Tresnawulansari, A. (2021). Optimasi Optical Density (Od) Pada Isolasi Genom Salmonella Typhi Menggunakan Genomic Dna Purification Kit. Jurnal Sains Dan Teknologi Farmasi Indonesia, 10(1), 35-43.
Setyawati, R., & Zubaidah, S. (2021). Optimasi konsentrasi primer dan suhu annealing dalam mendeteksi gen leptin pada sapi peranakan ongole (PO) menggunakan polymerase chain reaction (PCR). Indonesian Journal of Laboratory, 4(1), 36-40.
Silambi, A., Setyawati, T., & Langitan, A. (2020). Case report: Hirschsprung disease. Jurnal Medical Profession (MedPro), 2(1), 36-40.
Stujanna, E. N., Ningsih, S. S., Avissa, R., Ayu, N. P., Nurussofa, Z., Djuarna, D. J., ... & Sukarya, W. S. (2022). Collagen-VI Specific Primer Design Identification in Rats (Rattus norvegicus) Pancreas. Indonesian Journal of Medical Laboratory Science and Technology, 4(1), 60-70.
Tjan, A. (2021). Radiology perspective one-year study of Hirschsprung Disease. Folia Medica Indonesiana, 57(1), 41-45.
Violita, V., Achyar, A., Zulyusri, Z., Atifah, Y., Putri, D. H., & Nabilah, R. (2024). Primer Design and Optimization of Annealing Temperature for Gene Amplification GSTL2 on Rice. Al-Kauniyah: Jurnal Biologi, 17(2), 377-386.
Wei, F., Ge, Y., Li, W., Wang, X., & Chen, B. (2020). Role of endothelin receptor type B (EDNRB) in lung adenocarcinoma. Thoracic cancer, 11(7), 1885-1890.
Widowati, T., Melhem, S., Patria, S. Y., De Graaf, B. M., Sinke, R. J., Viel, M., ... & Sribudiani, Y. (2016). RET And EDNRB Mutation Screening in Patients with Hirschsprung Disease: Functional Studies and Its Implications for Genetic Counseling. European Journal of Human Genetics, 24(6), 823-829
Wu, T. T., Tsai, T. W., Chu, C. T., Lee, Z. F., Hung, C. M., Su, C. C., ... & Li, C. (2005). Low RET Mutation Frequency and Polymorphism Analysis of The RET And EDNRB Genes in Patients with Hirschsprung Disease In Taiwan. Journal Of Human Genetics, 50(4), 168-174.
Zheng, Y., Lan, C., Wang, N., Xu, X., Hu, T., Wu, Q., ... & Li, C. (2020). Significant Association of rs2147555 Genetic Polymorphism in the EDNRB Gene with Hirschsprung Disease in Southern Chinese Children. BioMed Research International, 2020(1), 5956412.
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Published
2025-05-29
How to Cite
Hidayah, N. ., Ahda, Y. ., Achyar, A. ., & Atifah, Y. . (2025). DESAIN PRIMER GEN EDNRB EKSON 4 DAN OPTIMASI PCR UNTUK ANALISIS MUTASI GEN PADA HIRSCHSPRUNG DISEASE. Jurnal Biogenerasi, 10(2), 1468–1476. https://doi.org/10.30605/biogenerasi.v10i2.5954
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