Optimized DNA Isolation Protocol from Pearl Millet (Pennisetum glaucum) Leaves with High Phenolic and Metabolite Content

Pearl millet (Pennisetum glaucum [L.] R. Br.) is a vital cereal crop recognized for its resilience to harsh environmental conditions. However, extracting high-quality DNA from pearl millet leaves presents a significant challenge due to the presence of phenolic compounds and secondary metabolites that interfere with molecular biology procedures. This study optimized a DNA isolation protocol tailored for pearl millet genotypes with high phenol and metabolite content to ensure the extraction of pure and intact DNA suitable for downstream molecular applications. Fresh leaf tissues from ten pearl millet genotypes with elevated phenolic content were processed using a modified cetyltrimethylammonium bromide (CTAB) protocol. The optimized method included key adjustments such as incorporating 1% polyvinylpyrrolidone (PVP) and increased concentration β-mercaptoethanol to neutralize phenolic compounds, alongside sodium chloride (1.4 M) to remove polysaccharides effectively. Sequential extractions with Phenol: chloroform: isoamyl alcohol and isopropanol precipitations were employed to purify DNA, while RNase treatment eliminated RNA contaminants. The resulting DNA exhibited high purity (A260/A280 ratios of 1.8–2.0) and integrity, suitable for applications including polymerase chain reaction (PCR)-based marker analysis was demonstrated. This optimized protocol offers a reliable and cost-effective approach for isolating high-quality genomic DNA from pearl millet, even from genotypes with high secondary metabolite content. It can facilitate genetic diversity studies, marker-assisted selection, and genomic sequencing, contributing to advancements in pearl millet breeding and research.