Quantitative and Qualitative Comparison of CTAB and SDS Method for DNA Extraction from the Herb Milk Thistle (Silybum marianum L.)

Document Type : Original Article

Authors

1 Associated Professor Biotechnology. Department of Biotechnology, Ramin University of Khuzestan, Molasani, Iran.

2 Bs of Genetic. Department of Genetic, Faculity of Welfare and Rehabilitation, Khuzestan.

3 Ph.D. oF Plant Breedin. Department of Plant Breedin at Zanjan University, Znjan, Iran.

4 ph.D student of Nanobiotechnology at I.H.University, Tehran, Iran

5 Department of Plant Pathology, Tarbiat Modares University, Tehran, Iran. Associated Professor Plant Pathology.

Abstract

Background and Objective: Quality of DNA is usually determined by multiple factors such as pollution especially caused by RNA, protein, lipid and other structures that inhibit Taq polymerase enzyme in polymerase chain reaction (PCR) and inhibit the cutting enzymes. The existence of phenolic compounds and other secondary metabolites has many effects on the quantity and quality of the purified DNA. At this investigation, in terms of quality and quantity with an efficient method for purification of DNA from S. marianum L. plant leaves, taking into account existing limitations to be provided.
Subjects and Methods: In this study quantitative and qualitative comparison between two DNA extraction methods namely sodium dodecyl sulfate (SDS) and a modified cetyl trimethylammonium bromide (CTAB),were undertaken. The quantity of extracted DNA was determined by measurement of the absorption rate in wavelengths of 260 and 280 nm and their ratios by spectrophotometer. For investigate DNA quality the existence of RNA fracture was evaluated by agarose gel.
Results: In terms of quantity and quality, for DNA extraction from S. marianum Populations, CTAB method had low performance While, SDS method showed appropriate performance in terms of quantity, quality and purity of extracted DNA.
Conclusion: ANOVA of samples revealed that the purification method, races and their interaction is very significant and showed that the purification of the races and their interaction had an impact on the quality of purified DNA. The 280A/260A ratio that is more than 2 indicates the presence of RNA and DNA degradation. Also if this ratio is less than 1.8 Indicating the presence of protein contamination in DNA. In genetic engineering researches and genetic studies, the suitable methods for DNA purifying are those that get higher quantity and quality of net DNA. Many studies carried out on medicinal plants and plants which contain high level of polyphenol and polysaccharide compounds showed that CTAB method and other methods that are technically same to CTAB have better efficiency. CTAB as a detergent can form complex with the DNA and can separate it from carbohydrates and proteins. The remaining polysaccharides are removed by concentrated NaCl.

Keywords

References

1-Aelayie, M, Naderi, R, Khalighi, A, Vazvayie, A, Salami, SG 2008, Study on effective components on the quantity and quality of genomic DNA extracted from Iran cyclamen, Pajouhesh Va Sazandgi journal, Vol.73, No.3, Pp.11-16.
2-Zamani, T, Sarkhosh, A, Fatahi Moghadam, MR, Ebadi, A 2007, Comparison of six genomic DNA extraction methods from Pomegranate (Punica granatum L.), Olom va Fonoon Baghebani Iran, Vol.6, No.2, Pp.99-110.
3-Shakibayie, MR 2005, Biotechnology and recombinant DNA, Moasesye Kerman Taksir, Kerman.
4-Kadkhodayie, S 2005, Simple and low-cost method for the isolation of nucleic acids from plants containing high polysaccharides and polyphenols: optimization of DNA extraction method in Almond, Third biotechnology congress of Iran, Pp.417-419.
5-Ahmad, R, Ferguson, L, Southwick, S 2003, Identification of pistachio (Pistacia vera L.) nuts with microsatellite markers. J. Amer. Soc. Hort. Sci., Vol.128, Pp.898-903.
6-Ahmadikhah, A 2009, A rapid mini-prep DNA extraction method in rice (Oryza sativa L.), African Journal of Biotechnology, Vol.8, No.2, Pp.323-327.
7-Bushra, C, Afshan, Y, Tayyab, H, Riazuddin, S 1999, Mini-scale genomic DNA extraction from cotton. Plant Mol. Bio. Rep., Vol.17, Pp.1-7.
8-Cheng, YJ, Guo, WW, Yi, HL, Pang, XM, Deng, X 2003, An efficient protocol for genomic DNA extraction from Citrus species. Plant Mol. Bio. Rep., Vol.21, Pp.177-177.
9-Fang, G,  Hammar, S, Grumet, R 1992, A quick and inexpensive method for removing poly saccharides from plant genomic DNA, Biotechniques, Vol.13, No.1, Pp.52-54.
10-Green, RL, Roinestad, IC, Boland, C, Hennessy, LK 2005, Developmental validation of the quantifiler real-time PCR kits for the quantification of human nuclear DNA samples, J. Forensic Sci., Vol.50, No.4, Pp.1-17.
11-Lodhi, MA, Ye, GN, Weeden, NF, Reisch, BI 1998, A simple and efficient method DNA extraction from grapevine cultivars and vitis species, Plant Mol. Bio.Rep.,Vol.12, No.1, Pp.6-13.
12-Loomis, MD 1974, Overcoming problems of phenolics and quinones in the isolation of plant enzymes and organelles. Methods Enzymol, Vol.31, Pp.528-544.
13-McCouch, SR 2002, A Brief Introduction to Rice DNA Technology, (http://www.ricelab.plbr.cornell.edu).
14-Poljak, M, Barlic, J, Seme, K, Avsic-Zupanc, T, Zore, A 1995, Isolation of DNA from archival Papanicolaou stained cytological smears using a simple salting-out procedure, J. Clin. Pathol., Vol.48, No.1, Pp.55-56.
15-Porebski, S, Bailey, LG, Baum, BR 1997, Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components. Plant Mol. Biol. Reptr., Vol.15, NO.1, Pp.8-15.
16-Shokrpour, M, Mohammadi, SA, Moghaddam, M, Ziai, SA, Javanshir, A 2008, Variation in flavonolignan concentration of milk thistle (Silybum marianum) fruits grown  in Iran, journal of herb, species & medicinal plants, Vol.13, No.4, Pp.55-69.
17-Suman, PS, Khanuja, Ajit, K, Shasany, MP, Darokar, S, Kumar, S 1999, Rapid Isolation of DNA from Dry and Fresh Samples of Plants Producing Large Amounts of Secondary Metabolites and Essential Oils. Plant Mol. Biol. Reptr., Vol.17, No.1, Pp.1-7.
18-Weishing, K, Nybom, H, Wolff, K, Meyer, W 1995, DNA isolation and purification. In: DNA fingerprinting in plants and fungi, Pp.44-59. CRC Press, Boca Raton, Florida.
Jundishapur Scientific Medical Journal
Volume 15, Issue 3 - Serial Number 102
July and August 2017
Pages 293-301

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History

  • Receive Date: 01 February 2016
  • Revise Date: 28 May 2016
  • Accept Date: 26 June 2016

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