Abstract
Extraction of non-degraded and contaminant-free DNA from field specimen requires collection under liquid nitrogen which is not readily available in resource constrained laboratories in low and middle income countries (LMICs). A method of extracting DNA from silica gel-preserved common bean (Proteus vulgaris L.) leaves is presented. The method, which does not involve the use of phenol, chloroform or isoamyl alcohol also obviates the need for low temperature incubation during the DNA extraction steps and the grinding of desiccated leaf tissue in liquid nitrogen. It relies on inactivating proteins using SDS and proteinase K along with precipitation of polysaccharides using a high salt solution (0.8 M NaCl). DNA is further purified by exploiting its insolubility in aqueous media. High quality pure DNA (mean concentration 2.84 ± 0.013 µg/g of dry leaf tissue) with mean DNA purity values of 2.1 ± 0.1 was extracted. The DNA was also found to be free of protein and polysaccharide contamination. This method enables DNA amplification using molecular markers routinely used in molecular biology laboratories like random amplified polymorphic (RAPD) markers, inter simple sequence repeat (ISSR) markers, sequence-characterized amplified region (SCAR) markers and simple sequence repeat (SSR) markers. The findings of this study show that it is possible to obtain high quality DNA from leaf tissue preserved in silica gel. The method used in this research will be invaluable to resource constrained laboratories in low and middle income countries (LMICs) that cannot afford to buy or access liquid nitrogen in order to extract high quality DNA and for research groups undertaking field surveys that require several days or weeks off station without laboratory freezers to maintain the integrity of the tissues which is crucial for obtaining high quality DNA. Key words: Random amplified polymorphic (RAPD), inter simple sequence repeat (ISSR), simple sequence repeat (SSR), sequence-characterized amplified region (SCAR), deoxyribonucleic acid (DNA), low and middle income countries (LMICs).
Highlights
Good quality of deoxyribonucleic acid (DNA) is a pre-requisite for most applications in a molecular biology laboratory
The DNA showed minimum degradation and it was amplified by all the molecular markers assayed (Figure 2)
DNA concentration was determined by substituting the values of the mean readings of fluorescence into the equation of the standard curve Y=0.11X+36 and multiplying the value by the volume of TE buffer which was used to dissolve the pellet (70 μl)
Summary
Good quality of DNA is a pre-requisite for most applications in a molecular biology laboratory. Of DNA mini prep protocols have been developed and some of them (Agbagwa et al, 2012; Sahu et al, 2012; Aubakirova et al, 2014; Abdel-Latif and Osman, 2017; Shu et al, 2018) rely on modification of earlier sodium dodecylsulphate (SDS) or cetyltrimethyl ammonium bromide (CTAB) protocols (Rogers and Bendich, 1985; Edwards et al, 1991). Extraction of DNA from plant tissues using in-house DNA extraction buffer formulations generally involves three stages; breaking the cell wall to release cellular constituents by grinding tissue in dry ice, liquid nitrogen or fine sand using a pestle and mortar or grinder; disrupting the cell membrane to release DNA into the extraction buffer; and use of detergents like SDS or CTAB to solubilize cell wall components and protect the DNA from nucleases using a chelating-like ethylenediaminetetracetic acid (EDTA). EDTA deprives the nucleases of magnesium ions thereby rendering them inactive
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