A Simple, Economical and Rapid Method to Isolate High Quality DNA from Oomycetes

Authors

  • PV Archana Central Tuber Crops Research Institute Sreekariyam, Thiruvananthapuram - 695017, India
  • Muthulakshmi Lajapathy Jeeva Central Tuber Crops Research Institute Sreekariyam, Thiruvananthapuram - 695017, India
  • Pravi V

Keywords:

Oomycetes, DNA extraction, YPT amplification, restriction analysis, RAPD

Abstract

Several approaches have been advocated to diagnose and control the devastating diseases caused  by the oomycetes, most of which are done using molecular techniques that require DNA with high  quantity and quality. An attempt has been made to develop simple, economical and rapid method toisolate high quality DNA from 10 different oomycetes at Central Tuber Crops Research Institute,  Thiruvananthapuram, India. This protocol was based on the sodium dodecyl sulphate (SDS) method,  which produced high quality DNA, either from liquid or plate cultures, without maceration in liquid  nitrogen, devoid of harmful chemicals like β.mercaptoethanol, phenol, chloroform, etc., and does  not require any incubation steps. The total time to complete the whole procedure was less than 30  minutes. The high quantity and purity of the isolated genomic DNA was confirmed by restriction  digestion analysis which demonstrated that the developed method provided DNA of sufficient qualityfor molecular analysis. The method developed is safe, convenient, economical and time saving like  commercial DNA extraction kits. It is useful for laboratories with minimum financial resources for  handling large number of samples .

Author Biographies

PV Archana, Central Tuber Crops Research Institute Sreekariyam, Thiruvananthapuram - 695017, India

Crop Protection , Ph. D student

Muthulakshmi Lajapathy Jeeva, Central Tuber Crops Research Institute Sreekariyam, Thiruvananthapuram - 695017, India

Indian Council of Agricultural Research,Principal Scientist

Pravi V

Central Tuber Crops Research InstituteSreekariyam, Thiruvananthapuram - 695017, India

References

Abad, Z. G., Palm, M., Shukl, R., Rice, S., Rascoe, J., Creswell, T., and Nelson, S. 2008. Morphological and molecular identification of eight putative new Phytophthora species from the USA. Third International Workshop Integration of Traditional and Modern Approaches for Investigating the Taxonomy and Evolution, Turin, Italy.

Buldewo, S., Jaufeerally-Fakim, Y. F. 2002. Isolation of clean and PCR amplifiable DNA from Anthurium andreanum. Plant Mol. Biol. Rep. 20: 71a-71g.

Chen Niu, Hirut Kebede1, Dick, L., Auld, Jason E., Woodward, Gloria Burow, and Robert J., Wright. 2008. Academic Journals Full Length Research Paper-A safe inexpensive method to isolate high quality plant and fungal DNA in an open laboratory environment. Afr. J. Biotechnol. Vol. 7 (16): 2818-2822.

Cooke, D. E. L., Drenth, A., Duncan, J. M., Wagels, G., and Brasier, C. M. 2000. A molecular phylogeny of Phytophthora and related Oomycetes. Fungal Genet. Biol. 30:17-32.

Duran, A., Slippers, B., Gryzenhout, M., Ahumada, R., Drenth, A., Wingfield, B. D., and Wingfield, M. J. 2009. DNA-based method for rapid identification of the pine pathogen, Phytophthora pinifolia. FEMS Microbiol. Lett. 298: 99-104.

Erwin, D. C. and Ribeiro, O. K. 1996. Phytophthora diseases worldwide. Amer. Phytopathol. Soc. Press, St. Paul, MN. 562 pp.

Hansen, T. V. O., Simonsen, M. K., Nielsen, F. C., Hundrup, Y. A. 2007. Collection of blood, saliva, and buccal cell samples in a pilot study on the Danish nurse cohort: comparison of the response rate and quality of genomic DNA. Cancer Epidemiol. Biomark Prev., 16: 2072.2076.

Horne, E. C., Kumpatla, S. P., Patterson, K. A., Gupta, M., Thompson, S. A. 2004. Improved high-throughput sunflower and cotton genomic DNA extraction and PCR fidelity. Plant Mol. Biol. Rep. 22: 83a-83i.

Karakousis, A., Tan, L., Ellis, D., Alexiou, H. 2006. An assessment of the efficiency of fungal DNA extraction methods for maximizing the detection of medically important fungi using PCR. J. Microbiol. Methods, 65: 38-48.

Kawabe, M., Mizutani, K., Yoshida, T., Teraoka, T., Yoneyama, K., Yamaguchi, I., Arie, T. 2004. Cloning of the pathogenicity-related gene FPD1 in Fusarium oxysporum f. sp. lycopersici. J Gen Plant Pathol. 70: 16.20.

Keb-Llanes, M., González, G., Chi-Manzanero, B., Infante, D. 2002. A rapid and simple method for small-scale DNA extraction in Agavaceae and other tropical plants. Plant Mol. Biol. Rep. 20: 299a-299e

Lamour K. and Finley L. 2006. A strategy for recovering high quality genomic DNA from a large number of Phytophthora isolates. Mycologia, 98:514.517

Li, H., Luo, J., Hemphill, J. K., Wang, J. T., Gould, J. H. 2001. A rapid and high yielding DNA miniprep for cotton (Gossypium spp.). Plant Mol. Biol. Rep. 19: 1-5.

Lugert, R., Schettler, C., and Gross, U. 2006. Comparison of different protocols for DNA preparation and PCR for the detection of fungal pathogens in vitro. Mycoses, 49: 298-304.

Mahuku, G. S. 2004. A simple extraction method suitable for PCR-based analysis of plant, fungal, and bacterial DNA. Plant Mol. Biol. Rep. 22: 71-81.

Sharma, K. K., Lavanya, M., Anjaiah, V., 2000. A Method for isolation and purification of peanut genomic DNA suitable for analytical applications. Plant Mol. Biol. Rep. 18: 393a-393h.

Sharma, R., Mahla, H. R., Mohapatr, T., Bhargava, S.C., 2003. Isolating plant genomic DNA without liquid nitrogen. Plant Mol. Biol. Rep. 21: 43-50.

Villa, N.O., Kageyama, K., Asano, T., Suga, H. 2006. Phylogenetic relationships of Pythium and Phytophthora species based on ITS rDNA, cytochrome oxidase II and b-tubulin gene sequences. Mycologia, 98: 410.422.

Zhang, D., Y. Yang, L.A. Castlebury and Cerniglia C.E. 1996. A method for the large scale isolation of transformation efficiency fungal genomic DNA. FEMS Microbiol Lett., 261-265.

Downloads

Published

2015-01-15

How to Cite

Archana, P., Jeeva, M. L., & V, P. (2015). A Simple, Economical and Rapid Method to Isolate High Quality DNA from Oomycetes. JOURNAL OF ROOT CROPS, 40(1), 80–84. Retrieved from https://ojs338.isrc.in/index.php/jrc/article/view/154

Issue

Section

Research Articles