Development of an efficient protocol for genomic DNA extraction from mango (Mangifera indica)

Dilruba Ashrafun Nahar Majumder, Lutful Hassan, Mohammad Abdur Rahim, Mohammad ahsanul Kabir

Abstract. Majumder DAN, Hassan L, Rahim MA, Kabir MA. 2011. Development of an efficient protocol for genomic DNA extraction from mango (Mangifera indica). Nusantara Bioscience 3: 105-111. A simple and efficient method for genomic DNA extraction from woody fruit crops containing high polysaccharide levels has been described here. In the present study, three kinds of plant DNA extraction protocols were studied and the target was to establish the water-saturated ether (WSE) with 1.25 M NaCl method as the most efficient protocol for removing the highly concentrated polysaccharides from genomic DNA of woody fruit crops. This method involves the modified CTAB or SDS procedure employing a purification step to remove polysaccharides using the WSE method. Precipitation with an equal volume of isopropanol caused a DNA pellet to form. After being washed with 70% ethyl alcohol, the pellet became easily dissolved in TE buffer. Using these three methods, DNA was extracted from samples of 60 mango genotypes, including young, mature, old, frosted old and withered old leaves. Compared with the three studied DNA extraction protocols of mango, it was found that the WSE method with NaCl had the highest value of average percentage (85.44%) in DNA content of the mango genotypes. The average yield of DNA ranged from 5.05 µg/µL to11.28 µg/µL. DNA was suitable for PCR and RAPD analyses and long-term storage for further use.

Key words: DNA extraction, fruit crops, polysaccharides, RAPD, water- saturated ether.

Abbreviations: CTAB: hexadecyltrimethylammonium bromide; RAPD: Random Amplified Polymorphic DNA; RFLP: Restriction Fragment Length Polymorphism; SSR: Simple Sequence Repeats; RT: Room temperature; WSE: Water: saturated ether.

Abstrak. Majumder DAN, Hassan L, Rahim MA, Kabir MA. 2011. Pengembangan protokol ekstraksi DNA genom mangga (Mangifera indica) yang efisien. Nusantara Bioscience 3: 105-111. Sebuah metode sederhana dan efisien untuk ekstraksi DNA genom tanaman buah berkayu yang mengandung banyak polisakarida telah dilakukan. Dalam penelitian ini, tiga protokol ekstraksi DNA tumbuhan dipelajari; dan tujuannya adalah menetapkan metoda ether jenuh air (WSE) dengan NaCl 1.25 M sebagai protokol yang paling efisien dalam mengeluarkan polisakarida yang sangat melimpah pada DNA genom tanaman buah berkayu. Penelitian ini mencakup CTAB yang dimodifikasi dan prosedur SDS sebagai langkah pemurnian untuk menghilangkan polisakarida, serta penggunaan metode WSE. Presipitasi dengan isopropanol yang sama volumenya menyebabkan pelet DNA terbentuk. Setelah dicuci dengan etil alkohol 70%, pelet menjadi mudah larut dalam buffer TE. Menggunakan tiga metode di atas, DNA diekstraksi dari sampel 60 genotipe mangga, termasuk daun muda, daun dewasa, daun tua, daun kering-beku dan daun kering. Perbandingan tiga protokol ekstraksi DNA mangga, menunjukkan bahwa metode WSE dengan NaCl menghasilkan nilai persentase rata-rata (85,44%) kandungan DNA genotipe mangga yang tertinggi. Hasil rata-rata DNA berkisar antara 5,05  µg/mL hingga 11,28  µg/mL. DNA cocok untuk analisis PCR dan RAPD dan memungkinkan penyimpanan jangka panjang untuk digunakan lebih lanjut.

Kata kunci: ekstraksi DNA, tanaman buah, polisakarida, RAPD, eter jenuh air.

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