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Comparison of methods for isolating high quality DNA and RNA from an oleaginous fungus Cunninghamella bainieri strain 2a1

Noor Adila A.K., and Farah Diba A.B., and Zamri Z.,, and Wan Mohtar W.Y., and Aidil A.H., and Mahadi N.M., and Murad A.M.A., (2007) Comparison of methods for isolating high quality DNA and RNA from an oleaginous fungus Cunninghamella bainieri strain 2a1. Malaysian Journal of Microbiology , 3 (1). pp. 7-13. ISSN 9834240708

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Official URL: http://www.usm.my/mjm/

Affiliations

Universiti Kebangsaan Malaysia. Faculty of Science and Technology. School of Biosciences and Biotechnology
Universiti Kebangsaan Malaysia. Faculty of Science and Technology. School of Biosciences and Biotechnology
Universiti Kebangsaan Malaysia. Faculty of Science and Technology. School of Biosciences and Biotechnology
Universiti Kebangsaan Malaysia. Faculty of Science and Technology. School of Biosciences and Biotechnology
Universiti Kebangsaan Malaysia. Faculty of Science and Technology. School of Biosciences and Biotechnology

Abstract

A number of protocols have been reported for efficient fungal DNA and RNA isolation. However, many of these methods
are often designed for certain groups or morphological forms of fungi and, in some cases, are species dependent. In this report, we compared four published protocols for DNA isolation from a locally isolated oleaginous fungus, Cunninghamella bainieri strain 2a1. These protocols either involved the use of polyvinyl pyrrolidone (PVP), hexacetyltrimethylammonium bromide (CTAB) or without using PVB or CTAB. For RNA isolation, we tested two published protocols, one of which is based on TRI REAGENT (Molecular Research Center, USA) and another is simple method employing phenol for RNA extraction and LiCl for precipitation. We found that the protocol involving the use of CTAB produced the highest genomic DNA yield with the best quality compared to other protocols. In the presence of CTAB, unwanted polysaccharides were removed and this method yielded an average amount of 816 ± 12.2 μg DNA/g mycelia with UV absorbance ratios A260/280 and A260/230 of 1.67 ± 0.64 and 1.97 ± 0.23, respectively. The genomic DNA isolated via this protocol is also suitable for PCR amplification and restriction enzyme digestion. As for RNA isolation, the method involving phenol extraction and LiCl precipitation produced the highest yield of RNA with an average amount of
372 ± 6.0 μg RNA/g mycelia. The RNA appears to be relatively pure since it has UV absorbance ratios A260/280 and A260/230 of 1.89 ± 2.00 and 1.99 ± 0.03, respectively. Finally, we have demonstrated that this method could produce RNA of sufficient quality for RT-PCR that amplified a 600 bp fragment of Δ12-fatty acid desaturase gene in C. bainieri.

Item Type:Journal
Additional Information:The authors would like to thank the financial support from the National Biotechnology Directorate, MOSTI for the financial support through grant no. 09-02-02-001 BTK/TD/001.
Keywords:Cunninghamella bainieri, DNA extraction, RNA extraction
Subjects:Q Science, Computer Science
T Technology, Engineering
ID Code:3521

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