As. Pac. J. Mol. Biol. & Biotech., Jan 2014 Vol. 1, 164-170
Sample preservation procedures and simple DNA isolation protocols for the tuberous crop, cassava (Manihot esculenta Crantz.)
Nuri Kiptantiyawati1, Nawar Lina Syarifah1, Pradita Maulia2, Etik Sulistiyowati1, Mirza Ramadhana Putra1, Yudiansyah1, Nurul Khumaida1, Sintho Wahyuning Ardie1*
1Department of Agronomy and Horticulture, Faculty of Agriculture, Bogor Agricultural University (IPB), Jl. Meranti Kampus IPB Darmaga 16680.
2Department of Biochemistry, Faculty of Mathematics and Natural Science, Bogor Agricultural University (IPB), Jl. Agatis Kampus IPB Darmaga 16680.
* Author for correspondence: Sintho Wahyuning Ardie
Department of Agronomy and Horticulture, Faculty of Agriculture, Bogor Agricultural University (IPB), Jl. Meranti, Kampus IPB Darmaga, Bogor, West Java, Indonesia 16680.
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Abstract.
Large scale collection of samples from remote areas followed by efficient DNA isolation is often required for genetic studies of a crop species using molecular methods. In this paper, we describe the comparison of different tissue preservation procedures, and develop a reliable and efficient protocol for isolating genomic DNA from four cassava (Manihot esculenta Crantz.) genotypes. We found that cassava leaf tissue can be preserved at 4°C for one week without significant loss in yield or quality. Comparison of six DNA isolation protocols revealed that a simplified version of an existing protocol using CTAB buffer was the best method to isolate DNA from leaf samples of ‘Ratim’, ‘Jame-jame’, ‘Malang-4’ and ‘Gajah’ genotypes. The methodology not only obtained high DNA yields of 771, 585, 542 and 2,672 μg DNA/g of sample, respectively, but also produced high quality DNA with A260/A280 ratios of 1.9, 1.9, 1.7 and 2.0, respectively. PCR amplification using the resulting DNA produced reliable and reproducible results, indicating the suitability of DNA for subsequent PCR analyses. This simplified protocol using CTAB buffer removes the necessity for grinding using liquid nitrogen and the use of phenol, PVP (polyvinyl polypyrrolidone), and β-mercaptoethanol in the isolation step, and can thus be considered technically easy and cost-effective.