International Journal of Scientific & Technology Research

Home About Us Scope Editorial Board Contact Us

IJSTR >> Volume 5 - Issue 9, September 2016 Edition

International Journal of Scientific & Technology Research  
International Journal of Scientific & Technology Research

Website: http://www.ijstr.org

ISSN 2277-8616

An Optimized DNA Extraction Protocol For Isolation Of High Quality Genomic DNA From Camphor Containing Timber Tree Species, Dryobalanops Beccarii Dyer

[Full Text]



Wei-Seng Ho, Kit-Siong Liew, Shek-Ling Pang



Genomic DNA, CTAB, Drynobanalops beccarii, Camphor, Secondary metabolites, PCR



Isolation of high-quality genomic DNA from Dryobalanops beccarii is obviously difficult due to the existence of large amounts of camphor and other secondary metabolites. These contaminants will co-precipitate with DNA during DNA isolation and purification processes, and therefore, resulting in a brownish DNA pellet that is unsuitable for downstream applications. Many DNA isolation protocols are available for various plant tissues; however these protocols are inefficient in yielding high-quality amplifiable genomic DNA especially from camphor containing timber tree species. A CTAB based protocol has been optimized for isolating genomic DNA from camphor containing timber tree species. Key steps include: 1) using 1% β-mercaptoethanol and 2% PVP 40 (Mr 40,000) in the extraction buffer; 2) sample incubation time, 40 minutes at 65C, and 3) DNA precipitation at room temperature (25C). The isolated DNA pellet was transparent colour and the purified genomic DNA is suitable for PCR amplification.



[1] W.S. Ho, S.L. Pang, P. Lau, & J. Ismail, (2011). Sequence variation in the cellulose synthase (SpCesA1) gene from Shorea parvifolia ssp. parvifolia mother trees. Journal of Tropical Agricultural Science 34(2): 323-329.

[2] W.S. Ho, A.S. Yii, S.M. Lee, T. Cheksum, I, Isa, S.L. Pang, & A. Julaihi, (2014). DNA Genotyping of Borneo Ironwood using M13 universal primer and SCAR marker development in Eusideroxylon zwageri. Acta Biologica Malaysiana 3(1): 10-15.

[3] P.S. Lai, W.S. Ho, & S.L. Pang, (2013). Development, characterization and cross-species transferability of expressed sequence tag-simple sequence repeat (EST-SSR) markers derived from Kelampayan tree transcriptome. Biotechnology 12(6): 225-235.

[4] K. Harrison, (2004). Camphor. Department of Chemistry, University of Oxford. Url: http://www.3dchem.com/molecules.asp?ID=203#

[5] E. Wickstrom, (1989). Camphor. International Programme on Chemical Safety (IPCS) INCHEM. Url: http://www.inchem.org/documents/pims/pharm/camphor.htm.

[6] I. Soerjanegara, & R.H.M.J. Lemmens, (1994). PROSEA 5 (1) Timber Trees: Major commercial timbers. PROSEA Foundation, Bogor, Indonesia. pp. 17-191.

[7] M. P. Shiva, & I. Jantan, (1998). Non-Timber Forest Products from Dipterocarps. In: Appanah, S. & Turnbull, J. M. (eds.) A review of Dipterocarp: Taxonomy, ecology and silviculture. pp. 192-193.

[8] W.D. Loomis, (1974). Overcoming problems of phenolics and quinones in the isolation of plant enzymes and organelles. Methods in Enzymology 31: 528-545.

[9] S. Porebski, L.G. Bailey, & B.R. Baum, (1997). Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components. Plant Molecular Biology Reporter 15(1): 8-15.

[10] W.S. Ho, R. Wickneswari, M.C. Mahani, & M.N. Shukor, (2004). The effects of the timing and method of logging on forest structure in Peninsular Malaysia. Forest Ecology and Management 203: 209-228.

[11] R.P. Adams, & J.E. Adams, (1992). Conservation of Plant Genes: DNA Banking and in vitro Biotechnology. Academic Press, INC, pp. 9-302.

[12] S.Y. Tiong, W.S. Ho, S.L. Pang, & J. Ismail, (2014). Nucleotide diversity and association genetics of xyloglucan endotransglycosylase/hydrolase (XTH) and cellulose synthase (CesA) genes in Neolamarckia cadamba. Journal of Biological Sciences 14(4): 267-375.

[13] J.J. Doyle, & J.L. Doyle, (1990). Isolation of plant DNA from fresh tissue. Focus 12: 13-15.

[14] M.G. Murray, & W.F. Thompson, (1980). Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res. 8: 4321-4325.

[15] E.T. Lau, W.S. Ho, & A. Julaihi, (2009). Molecular cloning of cellulose synthase gene, SpCesA1 from developing xylem of Shorea parvifolia spp. parvifolia. Biotechnology 8: 416-424.

A. Mischiels, W. Van den, M. Tucker, L. Van Riet, & A. Van Laere, (2003). Extraction of high-quality genomic DNA from latex-containing plants. Analytical Biochemistry 315: 85-89.

[16] D. Puchooa, (2004). A simple, rapid and efficient method for the extraction of genomic DNA from lychee (Litchi chinensis Sonn.). African Journal of Biotechnology 3(4): 253-255.

[17] R.A. Salzman, T. Fujita, K.Z. Salzman, P.M. Hasegawa, & R.A. Bressan, (1999). An improved RNA isolation method for plant tissues containing high levels of phenolic compounds carbohydrates. Plant Molecular Biology Reporter 17: 11-19.

[18] S.L. Pang, W.S. Ho, M.N. Mat-Isa, & Julaihi, A. (2015). Gene discovery in the developing xylem tissue of a tropical timber tree species: Neolamarckia cadamba (Roxb.) Bosser (Kelampayan). Tree Genetics & Genomes 11:47.

[19] M. Krizman, J. Jakse, D. Baricevic, B. Javornik, & M. Prosek, (2006). Robust CTAB-activated charcoal protocol for plant DNA extraction. Acta Agriculturae Slovenica 87: 427-433.