Extraction of High-quality Genomic DNA from Different Plant Orders Applying a Modified CTAB-Based Method | Chapter 07 | Modern Research in Botany Vol. 1
Background: Reliable measurement of DNA concentration and purity is important for almost all molecular genetics studies. Different plant species have varying levels of polysaccharides, polyphenols and other secondary metabolites which combine with nucleic acids during DNA isolation and further affect the quality of the extracted DNA. The current extraction protocol is based-upon the conventional CTAB method with further modifications for the extraction of DNA from variable plant seeds and crops belong to seven different orders The principle modifications currently employed for DNA extraction involved the use of higher CTAB concentration and higher levels of β-Mercaptoethanol. Additionally, higher concentrations of sodium chloride and potassium acetate were added simultaneously with absolute ice cold isopropanol for the precipitation of DNA free from polysaccharides.
Results and Conclusion: The prescribed modifications in the present method establish a quick and efficient standardized protocol for DNA extraction from different plant orders. The current extraction protocol, therefore, can be of great value for molecular analysis involving large numbers of different plant samples from different orders. These modifications consistently produced pure and high quality DNA suitable for further molecular analysis. Successful PCR amplification with RAPD primer, the complete digestion of the isolated DNA with the HindIII restriction enzyme and amplification of nptII gene applying both conventional and real-time PCR (RT-PCR) in presence of SYBR Green 1 dye pursued by the analysis of melting curve analysis validated the quality of the isolated DNA. Moreover, it reflects the efficiency of the protocol and proves its suitability for further applications for the assessment of food safety, detection of genetically modified (GM) crops and conservation of biodiversity.
Author(s) Details
Dr. Nadia Aboul-Ftooh Aboul-Maaty Department of Cell Biology, Genetic Engineering and Biotechnology Research Division, National Research Centre, Dokki, Cairo, Egypt.
Professor Hanaa Abdel-Sadek Oraby Department of Cell Biology, Genetic Engineering and Biotechnology Research Division, National Research Centre, Dokki, Cairo, Egypt.
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