Optimising DNA isolation for medicinal plants

Abstract

In African traditional health care systems medicinal plants have long been known to contain pharmacologically active compounds. This has led to an excessively high demand of these plant products resulting in the extinction of some plant species. With the application of molecular techniques in plant diversity conservation becoming increasingly popular, the isolation of PCR amplifiable genomic DNA becomes an important pre-requisite. However, medicinal plants are known to contain high levels of polyphenols and polysaccharides posing a major challenge in the isolation of high quality DNA. The objective of our research was to optimize a cetyl-trimethyl ammonium bromide (CTAB)-based protocol for the extraction of genomic DNA from a range of medicinal plant species, namely Sclerocarya birrea (tree), Barleria greenii (shrub), Aloe polyphylla and Huernia hystrix (both succulent plants). The quantity of DNA (µg/g) isolated using the modified CTAB protocol was higher for the lower plant tissue amounts (0.1 and 0.2 g) per 500 µl of extraction buffer. The spectral quality of DNA as measured by the A 260/ A 280 ratio ranged from 1.76 to 2.14 for S. birrea, B. greenii and A. polyphylla and 1.39 to 1.74 for H. hystrix. The DNA purity was further confirmed by restriction endonuclease digestion and PCR gel electrophoresis using operon arbitrary decamer primers (OPB-05, OPB-06 and OPG-07). The results show that the optimization of the amount of plant tissue per extraction buffer volume is a critical factor in genomic DNA isolation. In all cases the isolated DNA yielded high quantities from small amounts of plant tissue, and had good spectral qualities amenable to restriction endonuclease digestion and PCR amplification.