Vega Kartika Sari, Rudi Hari Murti


Perennial crop leaves contain polysaccharides, polyphenols, and other secondary metabolites in high concentration. The presence of those compounds inhibit enzymatic activities and amplification of DNA. The existing extraction methods were not able to dissociate the metabolites contaminants of sapodilla (Manilkara zapota (L.) van Royen) leaves and thus resulting in low quality of extracted DNA. The aim of this experiment was to develop an effective method to extract DNA from mature leaf samples of sapodilla (Manilkara zapota (L.) van Royen). Fifth modification of Doyle & Doyle DNA extraction protocol with modified concentration of buffer reagent (consisted of: CTAB 2.8%; NaCl 2.5M; mercaptoethanol 3%, and PVP 2.5%) and repetition of some phase purification (liquid nitrogen; three times CIAA; two times ethanol 70%, RNAse 1µl) generated high quality DNA and clear band of PCR amplification using RAPD primers.  




buffer modifications; DNA extraction; sapodilla

Full Text:



Amani, J., R. Kazemi, A.R. Abbasi and A.H. Salmanian 2011. A simple and rapid leaf genomic DNA extraction method for polymerase chain reaction analysis. Iranian J.Biotech. 9(1):69-71

Anuradha, H.J., K. Vijayan, C.V. Nair and A. Manjula. 2013. A novel and efficient protocol for the isolation of genomic DNA from mulberry (Morus L.). Emir. J. Food Agric. 25(2):124-131

Azmat, M.A., I.A. Khan, H.M.N. Cheema, I.A. Rajwana, A.S. Khan and A.A. Khan. 2012. Extraction of DNA suitable for PCR applications from mature leaves of Mangifera indica L. J. Zhejiang Univ-Sci. 13(4): 239-243

Chanda, S.V. and K. V. Nagani. 2010. Antioxidant capacity of manilkara zapota leaves extracts evaluated by four in vitro methods. Nature and Science. 8(10): 260-266

Chatrath, P., M. Choudhary and A. Tarafdar 2013. An efficient protocol for genomic DNA isolation from field grown mature leaves of Penniseteum gluacum. J.Biotech. 8(8):30-34

Chen, H., M. Rangasamy, S.Y. Tan, H. Wang and B.D. Siegfried. 2010. Evaluation of five methods for total DNA extraction from western corn rootworm beetles. Plos One. 5(8):1-6

Dehestani, A. and S.K.K. Tabar. 2007. A rapid efficient method for DNA isolation from plants with high levels of secondary metabolites. Asian J. Plant Sci. 6(6): 977-981

Doyle, J. 1991. DNA protocols for plants. In: Hewitt, G.M., A. W. B. Johnston, and J. P.W. Young. (Ed.). Molecular techniques in taxonomy. NATO ASI Series. Vol. 57. 283-293

Jose, J. and R. Usha. 2000. Extraction of geminiviral DNA from a high mucilaginous plant (Abelmoschus esculenthus). Plant Mol.Biol.Rep. 18:349-355

Niu, C., H. Kebede, D.L. Auld, J.E. Woodward, G. Burow and R.J. Wright. 2008. A safe inexpensive method to isolate high quality plant and fungal DNA in an open laboratory environment. African J. Biotech. 7(16):2818-2822

Ribeiro, R.A. and M.B. Lovato. 2007. Comparative analysis of different DNA extraction protocols in fresh and herbarium specimens of the genus Dalbergia. Genet. Mol. Res. 6:173-187

Sambrook, J., E.F. Fritsch and T. Maniatis. 1989. Molecular cloning: a laboratory manual. USA: Cold Spring harbor Lab Press.

Small, R.L., R.C. Cronn and J.F. Wendel. 2004. The use of nuclear genes for phylogeny reconstruction in plant. Aust. Syst. Bot. 17:145-170

Syafaruddin and T.J. Santoso. 2011. Optimation of DNA isolation and purification techniques on Reutalis trisperma (Blanco) Airy Shaw) (in Indonesian). J. Litri. 17(1): 11-17

Utami, A., R. Meryalita, N.A. Prihatin, L. Ambarsari, P.A. Kurniatin, and W. Nurcholis. 2012. Variation methods of DNA isolation from leaf of temulawak (Curcuma xanthorrhiza Roxb.) (in Indonesian). Proceedings of the National Conference of Chemistry. Unesa, Surabaya.pp.205-214

Vinod, K.K. 2004. Total genomic DNA extraction, quality check and quantitation. Tamil Nadu Agricultural University, Coimbatore. pp. 109-121


License URL: