Teknik Desain Primer untuk Amplifikasi Gen Tujuan Kloning dari DNA Agrobacterium tumefaciens
Primer Design Techniques for Gene Amplification Purpose of Cloning of DNA Agrobacterium tumefaciens
DOI:
https://doi.org/10.30605/perbal.v10i3.2090Keywords:
Agrobacterium tumefaciens, desain primer, DNA, PCRAbstract
Penelitian ini bertujuan untuk menghasilkan teknik desain primer untuk amplifikasi gen penyandi enzim dengan tujuan kloning dari DNA genom Agrobacterium tumefaciens. Metode pengumpulan informasi dari penelitian ini dilakukan dengan cara observasi langsung dan melakukan studi pustaka untuk menarik kesimpulan. Prosedur penelitian dimulai dari mengoleksi sekuen DNA genom Agrobacterium tumefasien dari gene bank (NCBI), desain primer menggunakan Genamics expression dan penambahan situs restriksi. Primer yang telah diperoleh dikonfirmasi melalui perangkat lunak past PCR dan amplifikasi gen dengan teknik polymerase chain reaction. Produk hasil amplifikasi dikonfirmasi menggunakan enzim restriksi SalI. Hasil penelitian yang telah diperoleh berupa poduk PCR berukuran 870 bp dan hasil konfirmasi menggunakan enzim SalI menghasilkan dua pita yaitu berukuran 359 bp dan 510 bp. Berdasarkan hasil yang diperoleh dapat disimpulkan bahwa teknik desain primer tujuan amplifikasi gen untuk kloning dapat dilakukan yaitu dengan kombinasi antara software dan melakukan penyisipan situs restriksi secara manual.
This study aims to produce a primer design technique for amplification of the gene encoding enzyme towards the cloning of gene from Agrobacterium tumefaciens. The genes information were collected from literatures study to decide the target gene and primer design. Methods: Firstly, we used genome sequences of the Agrobacterium tumefasien which is available on the gene bank (NCBI) to design a pears of primers, using Genemics expression, for amplifying the gene target. We also added restriction sites into the primers sequence. The specifity of primers designed then were evaluated by past PCR in silico software using the genome sequences as template and were confirmed by polymerase chain reaction amplification technique using the genome isolated from Agrobacterium tumefaciens as template. The targent gene was confirmed using the restriction enzyme SalI. Results: From Fast PCR amplification in silico, we obtained a single band with size of 870 bp, means that the primer designed were specific to the target gene. The PCR product has the same size with those of insilico study, and was digested with the SalI restriction enzyme, resulting two fragments with length of 359 bp and 510 bp, respectivelly. Conclusion: the amplificated genes was the target gene and the primers were successful designed.
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