Primer Design of EDNRB Gene Exon 5 and PCR Optimization for Gene Mutation Analysis in Hirschsprung's Disease
DOI:
10.29303/jbt.v25i2.8971Published:
2025-05-17Downloads
Abstract
HD disease is a common cause of neonatal intestinal obstruction. HD disease is a congenital disorder of the colon characterized by the absence of parasympathetic ganglion cells in the sub mucosal plexus of Meissneri and the mienterikus plexus of Auerbachi. Objective: To design specific primers for amplification of exon 5 in EDNRB gene and test the specificity in silico. To determine the optimum PCR conditions for amplification of exon 5 in EDNRB gene. Methods This type of research is descriptive. The purpose of descriptive research is to provide an overview or description of the EDNRB exon 5 gene primer design and PCR optimization. Electrophoresis results showed the temperature of 56 oC is the right temperature characterized by a single band and looks the thickest and brightest with the appropriate amplicons size of 367 bp, The results for the EDNRB gene primer exon 5 obtained the optimum concentration of 0.8 µM. This is characterized by DNA bands that look clear and thickest. The specific sequence of forward primer is 5'ATTTCA GAGACG GGAAG TGG3' reverse primer is 5'GGCACT CACTCATCATCTTG 3' which produces amplicons with a length of 367 bp. The optimum annealing temperature is 56 oC and the optimum primer concentration is 0.8 µM.
Keywords:
EDNRB gene, HD, primer design, optimization.References
Anissa, R. K., Lisdiana, L., & Widyayanti, A. T. (2023). Optimasi Metode Nested PCR untuk Deteksi Vibrio parahaemolyticus Ahpnd pada Udang Vaname (Litopenaeus vannamei). LenteraBio : Berkala Ilmiah Biologi, 13(1), 1–13. https://doi.org/10.26740/lenterabio.v13n1.p1-13
Amiel, J., Attié, T., Jan, D., Pelet, A., Edery, P., Bidaud, C., ... & Lyonnet, S. (1996). Heterozygous Endothelin Receptor B (EDNRB Mutations in Isolated Hirschsprung Disease. Human molecular genetics, 5(3), 355-357.
Anwar, M. A., Nurjanah, S. N., & Rahayu, W. P. (2022). Aplikasi basic local alignment search tool (BLAST) NCBI pada penelitian molekuler salmonella SPP. Syntax Literate ; Jurnal Ilmiah Indonesia,7(11),15446.https://doi.o rg/10 .364 18syntax-literate.v 7i11.9037
Chomdej, S., Leelawattanakul, P., Buddhachat, K., Pradit, W., Siengdee, P., Phongroop, K., & Nganvongpanit, K. (2018). ikinci. Kafkas Universitesi Veteriner Fakultesi Dergisi.https://doi.org/10.9775/kvfd.2018.20082
Dzikrina, H., Hidayat, T., & Sriyati, S. (2024). Pengembangan Primer Diagnostik Menggunakan Penanda mat-K Secara In Silico untuk Mendeteksi Kelangkaan Jenis Tumbuhan Di Indonesia. Al-Kauniyah:Jurnal Biologi, 17(1), 91-102.
Damayanti, F., Ramadhan, A. K., Nabil, A. A., Almuwaffaq, H. W., Smith, S., & Haikal, Z. (2023). Hirschsprung’s Disease: Metode Diagnosis dan Tatalaksana. Jurnal Ilmu Kedokteran dan Kesehatan, 10(12), 3584-3594.
Etskovitz, H., Kim, R. S., Wang, S. Z., & Nandivada, P. (2024). Surgical management of short-segment Hirschsprung disease. World Journal of Pediatric Surgery, 7(4), e000916.
Harahap, M. R. (2018). Elektroforesis: Analisis elektronika terhadap biokimia genetika. CIRCUIT: Jurnal Ilmiah Pendidikan Teknik Elektro, 2(1).
Karim, A., Tang, C. S.-M., & Tam, P. K.-H. (2021). The emerging genetic landscape of hirschsprung disease and its potential clinical applications. Frontiers in Pediatrics, 9, 638093. Htt ps://doi. org/10.3389/ fped.2021. 638093
Khorana, J., Phiromkanchanasak, P., Kumsattra, J., Klinoun, S., Aksorn, S., Chantakhow, S., Tepmalai, K., & Singhavejsakul, J. (2021). Development of clinical referral score model for early diagnosis of hirschsprung’s disease in suspected pediatric patients. Healthcare (Basel), 9(6).https://doi.org/10.3390/healthcare9060678
Khaira, A., Achyar, A., Zulyusri, Z., Atifah, Y., Putri, D. H., & Violita, V. (2023). Primer Design and Optimization of Annealing Temperature for Analysis of Glutathione Reductase Gene Expression in Rice (Oryza sativa L.). 3BIO: Journal of Biological Science, Technology and Management, 5(1), 142–148. https://doi.org/10.5614/3bio.2023.5.1.3
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. Borneo Journal of Medical Laboratory Technology, 6(1), 389–393. https://doi.org/1 0.33084/bjm lt.v6i1.6076
Palissei, A. S., Ahmadwirawan, A., & Faruk, M. (2021). Hirschsprung’s disease: epidemiology, diagnosis, and treatment in a retrospective hospital-based study. Journal of Thee Medical Sciences (Berkala Ilmu Kedokteran), 53(3).https://doi.org/10.19106/JMedSci005302202103
Setiadi, Q. H., & Haikal, Z. (2021). Total Colonic Aganglionosis: Dilema Diagnosis dan Dampak Jangka Panjang. Jurnal Kedokteran, 10(3), 531-536.
Sihotang, M. A., Erwinda, Y. E., Suwarni, E., & Lusianti, E. (2021). Desain Primer dan Analisis in Silico untuk Amplifikasi Gen mt-Co1 pada Tikus got (Rattus norvegicus). Eruditio: Indonesia Journal of Food and Drug Safety, 1(2), 20-29.
Sundari, S., & Priadi, B. (2019). Teknik isolasi dan elektroforesis DNA ikan tapah. Buletin Teknik Litkayasa Akuakultur, 17(2), 87-90.
Sari, E.N., R. W. Dewi , V. R. Dewi , S. C. Yowani , P. S. Yustiantara. (2018). Desain Primer untuk amplifikasi regio promoter gen inha isolat P016 multidrug resistance Mycobacterium Tuberculosis dengan metode polymerase chain reaction. Jurnal Farmasi Udayana, 7(1), 34-39.
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.
Salsabila, N., Fadilah, F., Pramana, R. C., Mutiatul K.A., S., Romzalis, A. A., Ramadhani, D. N., Rachmawati, Y., & Arianti, O. F. (2021). Penentuan Sekuens Terbaik untuk Gen COI pada Crocodylus rhombifer Menggunakan SoftWare Perlprimer dan Primer Blast Sebagai Bentuk Praktikum Saat Pandemi Covid-19. Indonesian Journal of Science Learning (IJSL), 2(1), 15–21. https://doi.org/10.15642 /ijsl.v2i1.1233
Torroglosa, A., Villalba-Benito, L., Luzon-Toro, B., Fernández, R. M., Antiñolo, G., & Borrego, S. (2019). Epigenetic mechanisms in hirschsprung disease. International Journal of Molecular Sciences, 20(13). https://doi. org/10.3390/ijms20133123
Wijayana, K. A. (2023). Penyakit Hirschsprung: Sebuah Tinjauan Pustaka Tentang Patofisiologi, Terapi. Mandala Of Health, 16(2), 134-145.
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. https://doi.org/10.1111/1759- 7714.13474
Xiao, J., Hao, L.-W., Wang, J., Yu, X.-S., You, J.-Y., Li, Z.-J., Mao, H.-D., Meng, X.-Y., & Feng, J.-X. (2023). Comprehensive characterization of the genetic landscape of familial Hirschsprung’s disease. World Journal of Pediatrics, 19(7), 644–651. https://doi.o rg/10.1007/s1 2519-023-00686-x
Yuenleni, Y. (2019). Langkah-Langkah Optimasi PCR. Indonesian Journal of Labo ratory, 1(3), 51-56.
Yustinadewi, P. D., Yustiantara, P. S., & Narayani, I. (2018). Teknik perancangan primer untuk sekuen gen MDR-1 varian 1199 pada sampel buffy coat pasien anak dengan LLA. Jurnal metamorfosa, 5(1), 105-111.
License
Copyright (c) 2025 Septiasri Anggun, Yuni Ahda
This work is licensed under a Creative Commons Attribution 4.0 International License.
Jurnal Biologi Tropis is licensed under a Creative Commons Attribution 4.0 International License.
The copyright of the received article shall be assigned to the author as the owner of the paper. The intended copyright includes the right to publish the article in various forms (including reprints). The journal maintains the publishing rights to the published articles.
Authors are permitted to disseminate published articles by sharing the link/DOI of the article at the journal. Authors are allowed to use their articles for any legal purposes deemed necessary without written permission from the journal with an acknowledgment of initial publication to this journal.
