Comparison of Two PCR Primer Sets for In-House Validation of GHSR Gene Variation Detection Employing Artificial Recombinant Plasmid Approach

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Published: May 29, 2024
DOI: https://doi.org/10.19109/biota.v10i2.21166
Keywords:
DNA variant; GHSR gene; Primer set, PCR-sequencing; Stunting

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Ahsanal Kasasiah
Department of Pharmacy, Faculty of Health Science, University of Singaperbangsa Karawang, West Java, Indonesia
Jekmal Malau
Department of Pharmacy, Faculty of Health Science, University of Singaperbangsa Karawang, West Java, Indonesia
Sekar Andjung Tresnawati
The National Population and Family Planning Board for West Java, West Java, Indonesia
Priscinya Christiana Debora
Department of Pharmacy, Faculty of Health Science, University of Singaperbangsa Karawang, West Java, Indonesia
Nur Komala Fitri
Department of Pharmacy, Faculty of Health Science, University of Singaperbangsa Karawang, West Java, Indonesia
Saarah Hamidah Asmara Indratno
Department of Pharmacy, Faculty of Health Science, University of Singaperbangsa Karawang, West Java, Indonesia
Asman Hitopik
Department of Pharmacy, Faculty of Health Science, University of Singaperbangsa Karawang, West Java, Indonesia
Eriyanti Astika
Department of Pharmacy, Faculty of Health Science, University of Singaperbangsa Karawang, West Java, Indonesia
Anisa Aula Rahma
Department of Pharmacy, Faculty of Health Science, University of Singaperbangsa Karawang, West Java, Indonesia
Al Mukhlas Fikri
Department of Nutrition, Faculty of Health Science, University of Singaperbangsa Karawang, West Java, Indonesia

Abstract

Stunting is a significant global public health problem caused by long-term dietary deficits that affect many children worldwide. Both environmental and genetic factors, including variants in the GHSR gene, play a crucial role in stunted growth. This study used an artificial recombinant plasmid DNA method to evaluate two primer set combinations for identifying DNA variants in the GHSR gene. Selecting suitable primer sets for identifying GHSR genetic variants linked to stunting is essential, as evidenced by PCR and sequencing techniques. The target gene, based on the GHSR reference sequence, consists of eight DNA variations (ΔQ36, G57G, P108L, L118L, R159R, C173R, D246A, and A277P). A recombinant plasmid was created by inserting a 1000 bp fragment of the GHSR gene into the pUC57 backbone. Primer sets were chosen based on their capacity to amplify these eight genetic variations and were optimized and validated using PCR methods. PCR and bi-directional sequencing verified the existence of surrounding DNA and specific single nucleotide variants (SNVs). In our study, we discovered four changes in the DNA sequence (R159R G>A, C173R T>C, D246A A>C, and A277P G>C) using the E1_F2/E1_R3 primer pair. Additionally, a new combination of primers (E1_F1/E1_R3) effectively detected seven DNA sequence mutations (ΔQ36 del CAG, G57G C>T, P108L C>T, L118L C>T, R159R G>A, C173R T>C, and D246A A>C). We have developed a new combination of forward and reverse primers to identify seven SNVs in the GHSR gene, which could serve as a diagnostic tool in clinical laboratory settings.

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Comparison of Two PCR Primer Sets for In-House Validation of GHSR Gene Variation Detection Employing Artificial Recombinant Plasmid Approach. (2024). Jurnal Biota, 10(2), 128-143. https://doi.org/10.19109/biota.v10i2.21166
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Vol. 10 No. 2 (2024): Jurnal Biota 2024
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How to Cite

Comparison of Two PCR Primer Sets for In-House Validation of GHSR Gene Variation Detection Employing Artificial Recombinant Plasmid Approach. (2024). Jurnal Biota, 10(2), 128-143. https://doi.org/10.19109/biota.v10i2.21166

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