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<p>The growing integration of advanced technologies in molecular studies has highlighted the need for efficient and reliable DNA extraction methods, as the accuracy and success of these technologies are highly dependent on DNA purity and integrity. Efficient isolation of high-quality genomic DNA is a prerequisite for next-generation sequencing (NGS) and other molecular applications, yet remains challenging in woody species owing to high levels of phenolics, polysaccharides, and secondary metabolites. The present study aimed to optimize a reliable DNA extraction protocol for <i>B. wallichiana</i>. Five different protocols – conventional CTAB (CCP), activated charcoal (ACP), modified CTAB (MCP), modified activated charcoal (MACP), and double buffer protocol (DBP) were evaluated using leaf tissues. The comparative analysis using various parameters such as DNA yield, purity, integrity, and storage stability demonstrated that MCP was most effective. Notably, this protocol consistently produced higher yields (301 to 850 ng/µL) with stable purity (A<sub>260/280</sub> = 1.82–2.01), and intact DNA, outperformed other methods that showed variability, contamination, and degradation. Further validation through polymerase chain reaction using five Inter Simple Sequence Repeat (ISSR) primers produced clear and reproducible amplification, confirming that MCP is suitable for downstream genetic analysis. The study provides a reliable, efficient, and cost-effective approach for molecular characterization of <i>B. wallichiana</i> and will facilitate broader application in conservation genetics of Himalayan woody species rich in secondary metabolites and phenolics. By enabling stable yield and purity of genomic DNA, the protocol will contribute in developing informed conservation strategies and support sustainable management of its genetic resources.</p>

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Optimization of a high-yield genomic DNA isolation protocol for Buxus wallichiana Baill., an endemic species of the Northwestern Himalaya

  • 期刊论文

  • 发表日期：

    2026年7月18日

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  • Haripriya Kavidayal,
  • Aman Dabral,
  • Shivam Kishwan,
  • Harish Singh Ginwal,
  • Hukum Singh

摘要

The growing integration of advanced technologies in molecular studies has highlighted the need for efficient and reliable DNA extraction methods, as the accuracy and success of these technologies are highly dependent on DNA purity and integrity. Efficient isolation of high-quality genomic DNA is a prerequisite for next-generation sequencing (NGS) and other molecular applications, yet remains challenging in woody species owing to high levels of phenolics, polysaccharides, and secondary metabolites. The present study aimed to optimize a reliable DNA extraction protocol for B. wallichiana. Five different protocols – conventional CTAB (CCP), activated charcoal (ACP), modified CTAB (MCP), modified activated charcoal (MACP), and double buffer protocol (DBP) were evaluated using leaf tissues. The comparative analysis using various parameters such as DNA yield, purity, integrity, and storage stability demonstrated that MCP was most effective. Notably, this protocol consistently produced higher yields (301 to 850 ng/µL) with stable purity (A260/280 = 1.82–2.01), and intact DNA, outperformed other methods that showed variability, contamination, and degradation. Further validation through polymerase chain reaction using five Inter Simple Sequence Repeat (ISSR) primers produced clear and reproducible amplification, confirming that MCP is suitable for downstream genetic analysis. The study provides a reliable, efficient, and cost-effective approach for molecular characterization of B. wallichiana and will facilitate broader application in conservation genetics of Himalayan woody species rich in secondary metabolites and phenolics. By enabling stable yield and purity of genomic DNA, the protocol will contribute in developing informed conservation strategies and support sustainable management of its genetic resources.