Comparative analysis of different methods of Hedera helix DNA extraction and molecular evidence of the functionality in PCR

Abstract

Received: 2016-06-25 | Accepted: 2016-07-29 | Available online: 2016-12-22 http://dx.doi.org/10.15414/afz.2016.19.04.144-149 The most suitable method of total DNA extraction still remains the crucial step for many plant species, although there are many different protocols and commercial kits for DNA isolation. In this study, five different extraction protocols were analysed to find out the most appropriate method for DNA extraction from Hedera helix L. This species has numerous medical and pharmaceutical uses and is also characterized by antioxidant effects on human body. In spite of its wide medical utilization, it belongs to those plant species, where the genomic information is very limited. Comparing of different protocols resulted in the yield of extracted DNA that has ranged from 6.3 to 487 ng μl-1. The purity of extracted DNA has ranged from 1.4 up to 2.0 A260/A280. All the extraction methods used in this study were evaluated not only in term of quantity and purity of DNA but also its functionality in the restriction endonuclease digestion and polymerase chain reaction based downstream analysis was performed. Keywords: DNA extraction, CTAB, SDS,commercial kit, Hedera helix L. References ABU-ROMMAN, S. (2011). Comparison of methods for isolating high quality DNA from sage (Salvia officinalis). Journal of Medicinal Plants Research, vol 5, no. 6, pp. 938-941 BALANSARD, G. et al. (1980). Douvicidal and Antifungal Activities of α-Hederin Extracted from Hedera helix leaves. Planta Medica, vol. 39, p. 234. BARRA, M. et al. (2012). Simple and robust DNA extraction mehod for the large-scale analysis of genotypes containing high polyphenolic content, such as landraces of Solanum tuberosum and Zea mays. Plant breeding, genetic and genetic resources, vol. 39, no. 3, pp. 593-601. BEDIR, E. et al. (2000). Triterpene Saponins from the Fruits of Hedera helix. Phytochemistry, vol. 53, pp. 905-909. CLARKE, M.M. et al., (2006). Prevalence of different horticultural taxa of ivy (Hedera spp., Araliaceae) in invading populations. Biological Invasions, vol. 8, pp. 149–157. COTA-SÁNCHEZ, H. J. et al. (2006). Ready-to-Use DNA Extracted with a CTAB Method Adapted for Herbarium Specimens and Mucilaginous Plant Tissue. Plant Molecular Biology Reporter, vol. 24, no. 2, pp.161-167. CRESPIN, F. et al. (1995). Identification of 3O-beta-D-Glucopyranosyl Hederagenin from the Leaves of Hedera helix. Fitoterapia, vol. 66, pp. 47. DABO, S. M. et al. (1993). A method for the isolation of nuclear DNA from cotton (Gossypium) leaves. Analytical Biochemistry, vol. 210, pp. 34-38. DELLAPORTA, S. L. et al. (1983). A Plant DNA Minipreparation: Version II. Plant Mol. Biol. Rep., vol. 1, pp. 19-21. DEMEKE, T. and JENKINS, R. (2010). Influence of DNA extraction methods, PCR inhibitors and quantification methods on Real time PCR assay of biotechnology-derived traits. Analytical and Bioanalytical Chemistry, vol. 396, pp. 1977-1990. DOOSTY, B. et al. (2012). Comparative Analysis and Optimization of Different DNA Extraction Protocols in Satureja khuzistanica. International Journal of Biology, vol. 4, no. 4,pp. 112-116. ELIAS, R. et al. (1991). Triterpenoid saponins from the leaves of Hedera helix. Journal of Natural Products, vol. 54, pp. 98-103. GREEN, A. F. et al. (2011). Phylogeny and Biogeography of Ivies ( Hedera spp., Araliaceae), a Polyploid Complex of Woody Vines. Systematic Botany, vol 36, no. 4, pp. 1114-1127. HUAQIANG, T. et al. (2013). Comparative Analysis of Six DNA Extraction Methods in Cowpea (Vigna unguiculata L.Walp). Journal of Agricultural Science, vol 5, pp. 82-90. IVERSEN, J. (1944). Viscum, Hedera and Ilex as climatic indicators. Geologiska Föreningers Stockholm Förhandlinger, vol. 66, pp. 463–483. JOBES, D. V. et al. (1995) Plant DNA isolation: A Method to Efficiently Remove Polyphenolics, Polysaccharides, and RNA. Taxon, vol. 44, pp. 379-386. KALENDAR, R. et al. 2010. iPBS: a universal method for DNA Fingerprinting and retrotransposon isolation. Theoretical and Applied Genetics, vol. 121, pp. 1419–1430. KRAJEWSKA, A. (1981). Phytochemical Studies of Hedera helix Herbage, Flowers and Fruits. Herba Polonica, vol. 27, pp. 303-309. LADE, B.D. et al., (2014). Efficient genomic DNA extraction protocol from medicinal rich Passiflora foetida containing high level f polysacharide and polyphenol. SpingerPlus, vol. 3, p. 457. LUTSENKO, Y. et al. (2010). Hedera helix as a medicinal plant. Herba Polonica, vol. 56, pp. 83- 96. MAJESTERSAVORNIN, B. et al. (1991). Saponins of the ivy plant. Planta Medica, vol. 57, p. 260- 262.  Â