Polymerase Chain Reaction (PCR) amplification of genomic DNA is a crucial step for molecular studies such as sequencing and genotyping because it requires pure and high-quality DNA. However, the presence of secondary metabolites characteristic of plants with pharmaceutical properties makes plant DNA isolation a limiting factor in molecular biology research and genomic studies. Therefore, it is necessary to establish an efficient protocol to eliminate these compounds, as they are the main agents interfering with plant genomic DNA isolation and amplification processes. In this study, we compared five different DNA extraction protocols with the commonly known cashew (Anacardium occidentale L.,). Among these protocols are the Zymo Research Kit, the standard CTAB method [1], the sodium bisulfite-based Porebski protocol [2], the CTAB method described in [3] employing sorbitol, and a customized CTAB protocol. We evaluated the performance of each protocol in terms of DNA yield and quality, with the aim of determining their effectiveness in removing secondary metabolites including polyphenols, proteins, and polysaccharides, and assessing how these factors impact PCR amplification. By modifying the original CTAB method, by reduction of the iniatial leaf amount to from 100 mg to around 20 - 50 mg and increase of the concentration of antioxidant DTT from the 1% to 2%, we achieved efficient removal of secondary metabolites, resulting in good yields of high-quality DNA concentration (259.71 ng/μl), excellent purity (1.78), and the highest success rate (88%) of PCR test. Clear bands of DNA were obtained by visualization on 1% agarose gel, indicating the effectiveness of this method in suppressing the inhibitory effects of secondary metabolites on the enzymes used in molecular studies. Overall, the comparative analysis shows that while several protocols produced acceptable DNA concentrations and purity levels, only the Modified CTAB protocol successfully eliminated enough secondary metabolites to allow PCR amplification.
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