Abstract
Yam (Dioscorea species) and banana (Musa species) leaf sample contain high levels of polysaccharide and polyphenolic compounds. Extraction of good quality DNA from such leaves is usually problematic. Therefore, there is a need to extract good quality DNA in order to perform downstream DNA analysis, especially for the detection of viruses due to their low titration in some infected leaves. The cetyltrimethylammonium bromide (CTAB) DNA extraction method was selected in order to optimise DNA extraction from plant material with high level of polysaccharides. Four steps of the Lodhi et al. (1994) method were modified to be user friendly in less resource laboratories of developing countries. Yield and purity of the extracted DNAs were quantified using a NanoDrop 2000 spectrophotometer and by migration on an agarose gel after polymerase chain reaction (PCR) amplification. The results of the DNA yields and purity were in the range of 287.40±2.23 to 424.95±1.85 ng/ul and 2.10±0.05 to 2.19±0.04, respectively. Modification was found to yield DNA of reasonable quantity, quality and purity. Furthermore, the method can be used in a laboratory with less sophisticated waste disposal system. Key words: Deoxyribonucleic acid (DNA), cetyltrimethyl ammonium bromide (CTAB), yam, banana, tomato.
Highlights
Yam (Dioscorea species) leaf sample contain high levels of polysaccharide and polyphenolic compounds
Plant samples used for the modification of the cetyltrimethylammonium bromide (CTAB) method and detection of badnaviruses consisted of fresh yam leaf samples obtained from the International Institute of Tropical Agriculture (IITA) (n = 231), dried yam leaf samples collected by Dr Ed Canning in 1997 (n = 44) during Cameroon survey and Professor Susan Seal in 2001 (n = 7) from CIRAD France, as well as fresh and etiolated leaves of Dioscorea rotundata, Dioscorea alata, banana and tomato grown in the quarantine glasshouse at Natural Resources Institution, UK
The NS step was incorporated into the Lodhi et al (1994) method to reduce the oxidation of phenolic plant metabolites during Deoxyribonucleic acid (DNA) extraction
Summary
Yam (Dioscorea species) leaf sample contain high levels of polysaccharide and polyphenolic compounds. The presences of these compounds in yam leaf tissue often affect the isolation of good quality DNA (Kenyon et al., 2008). This has presented a problem for the downstream molecular analyses of extracted DNAs from yam samples especially in the detection and analysis of yam viruses such as badnaviruses. Polymerase chain reaction (PCR) methods were used to evaluate DNA purity by detecting the presence of yam badnavirus sequences on samples known to be badnavirus positive. PCR techniques provide greater sensitivity compared to other methods such as symptom observation and serological methods (Kenyon et al, 2008; Seal et al, 2014)
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