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Detection of Transgene in Tilapia (oreochromis Niloticus) by Polymerase Chain Reaction (pcr) Technique

P. C. Ray
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh- 2202.

M. S. Alam
Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh- 2202.

M. S. Islam
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh- 2202.

Abstract/ Executive Summary:

Polymerase Chain Reaction (PCR) technique using specific primer can be used to detect transgenes. The present study was undertaken to detect salmon growth hormone (GH) gene in transgenic tilapia (Oreochromis niloticus) by PCR. DNA was extracted from F1 Tilapia generated by crossing transgenic parents. Two primers were designed to amplify a part of the region of GH gene sequence, which was used to make transgenic tilapia. To confirm the specificity of the selected primer, PCR was performed on diluted DNAs, extracted from tilapia fin tissues. GH transgene sequences (1500 bp) were successfully amplified from transgenic fish in this study. The specificity of the primers was found to be high in detecting the salmon GH transgenes. The PCR-based method therefore, could be used for fast and easy screening of transgenic fish for this gene.

Keywords: Growth hormone (GH) gene, PCR amplification, Transgenic tilapia

Location: Department of Fisheries Biology and Genetics and Professor Mohammad Hossain’s Central Laboratory, Bangladesh Agricultural University, Mymensingh.

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Tilapia

Objectives:

To detect salmon growth hormone (GH) transgenes in tilapia (O. niloticus) using PCR technique.

Methodology:

Fingerlings of transgenic tilapia (O. niloticus) were collected from the University of Southampton, U.K. in January 2006 and grown in aquarium in the Department of Fisheries Biology and Genetics. The experiment was carried out at the Department of Fisheries Biology and Genetics and Professor Mohammad Hossain’s Central Laboratory, Bangladesh Agricultural University, Mymensingh. Genomic DNA was isolated from fin tissues of fish. Fin tissues were collected from 12 fishes and stored separately in 95% ethanol at room temperature. DNA was isolated from these samples by the phenol-chloroform extraction and ethanol precipitation method. In brief, approximately 20 mg of fin tissues were cut into small pieces and taken into 1.5 ml microcentrifuge tube containing extraction buffer (100mM Tris-HCl, 10 mM EDTA, 250 mM NaCl, pH 8.0 and 1% SDS) and proteinase-K (10 mg/ml). After homogenization, samples were kept at 37°C overnight. Then phenol, chloroform and isoamylalcohol (25:24:1, v/v/v) purification and ethanol precipitation was performed. DNAs pellets were diluted in TE (10 mM Tris-HCl and 1 mM EDTA, pH 8.0) and stored at -20°C. Isolated DNAs were confirmed by running on 0.8% agarose gel and quantified by using a spectrophotometer (Spectronic ^® Genesys ^TM ). PCR analysis was performed using primers P 1 (5´-ACCTGTGGAGACTGTTGAGAT-3´) and P 2 (5´-CTACTTAGACCACTCAATTGG -3´) to detect GH transgene. Since these primers amplify salmon GH transgene fragment only, this reduces the chances of obtaining false positive results from any endogenous fish DNA sequences. PCR reactions were performed on each DNA sample in a 10µl reaction mix containing 1µl of 10x ampli Taq polymerase buffer, 2.5 µl of each of primers P₁ and P₂ (10µM each), 1µl of dNTPs (250µM each), 2 µl of template DNA (25ng/µl), 0.2µl of ampli Taq DNA polymerase (1 unit, Genei, Bangalore, India) and 0.8 µl of sterile water. DNA amplification was performed in an oil-free thermal cycler (Master cycler Gradient, Eppendorf, Germany). The reaction mix was preheated at 95°C for 3 min denaturation followed by 30 cycles of 1 min at 95°C, annealing at 59°C for 1 min and elongation or extension at 72°C for 2 min. After the last cycle, a final step of 5 min at 72°C was added to allow complete extension of all amplified fragments. After completion of cycling program, reactions were held at 4°C. PCR products were run on 1% agarose gel at 120 volt for about 1 hr 30 min. Molecular weight markers markers (λ DNA HindIII digest, Thermo Fisher Scientific and pUC18/Taq I digest, Genei, Bangalore, India) were run alongside the gel to ensure the size of PCR products. The gel was observed under UV light on a transilluminator and photographed by a BioDoc-It^TM system.

Source of Information: J. Environ. Sci. & Natural Resources, 8(2): 47-49, 2015, ISSN 1999-7361

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

PCR has been provided to be a powerful tool for detection of transgens in transgenic fish. This could be used as a baseline study for future transgenes detection in transgenic plants, animals, fish or other organisms in Bangladesh.The specificity of the primers was found to be high in detecting the salmon GH transgenes. The PCR-based method therefore, could be used for fast and easy screening of transgenic fish for this gene.

Knowledge Management: Journal