Gill Komaljeet, Negi Shivanti, Kumar Pankaj, Irfan Mohammad
Department of Biotechnology, Dr. Yashwant Singh Parmar University of Horticulture and Forestry, Solan, Himachal Pradesh, India.
Plant Biology Section, School of Integrative Plant Science, Cornell University, Ithaca, NY, USA.
Mol Biol Rep. 2025 Jun 26;52(1):638. doi: 10.1007/s11033-025-10763-1.
Citrus species are recalcitrant to DNA extraction due to high levels of polysaccharides, polyphenols, and secondary metabolites that interfere with DNA yield and purity. This study presents an optimized cetyltrimethylammonium bromide (CTAB)-based protocol for efficient genomic DNA isolation from citrus leaves.
Leaf tissues were ground in liquid nitrogen and extracted using a CTAB buffer supplemented with 2% β-mercaptoethanol and 2% polyvinylpyrrolidone. Contaminants were removed by two rounds of phenol: chloroform: isoamyl alcohol (25:24:1) extraction. RNase A treatment was applied, followed by DNA precipitation using cold isopropanol. DNA quality was assessed by A260/280 absorbance ratios and agarose gel electrophoresis. Statistical comparisons were made using the Mann-Whitney U test, Cohen's d, and bootstrap resampling.
The modified protocol yielded high-molecular-weight DNA with A260/280 ratios ranging from 1.78 to 1.98, indicating high purity. Electrophoresis confirmed intact DNA with minimal RNA contamination. DNA yield and band intensity were significantly higher than with the conventional method (p < 0.001, Cohen's d = -2.10). A positive correlation (r = 0.23) was observed between yield and band intensity. Bootstrap confidence intervals for mean band intensity were 0.11-0.20 (conventional) and 2.91-4.84 (modified).
The optimized CTAB protocol provides a simple, cost-effective, and reproducible method for high-quality genomic DNA extraction from citrus, suitable for PCR, genotyping, and sequencing applications.
由于高水平的多糖、多酚和次生代谢物会干扰DNA产量和纯度，柑橘类物种的DNA提取具有挑战性。本研究提出了一种基于十六烷基三甲基溴化铵（CTAB）的优化方案，用于从柑橘叶片中高效分离基因组DNA。
将叶片组织在液氮中研磨，并用补充了2%β-巯基乙醇和2%聚乙烯吡咯烷酮的CTAB缓冲液进行提取。通过两轮苯酚：氯仿：异戊醇（25:24:1）提取去除污染物。进行核糖核酸酶A处理，然后用冷异丙醇沉淀DNA。通过A260/280吸光度比值和琼脂糖凝胶电泳评估DNA质量。使用曼-惠特尼U检验、科恩d值和自助重采样进行统计比较。
改进后的方案产生了高分子量DNA，A260/280比值在1.78至1.98之间，表明纯度高。电泳证实DNA完整，RNA污染最小。DNA产量和条带强度显著高于传统方法（p < 0.001，科恩d值 = -2.10）。产量与条带强度之间观察到正相关（r = 0.23）。平均条带强度的自助置信区间为0.11 - 0.20（传统方法）和2.91 - 4.84（改进方法）。
优化的CTAB方案为从柑橘中提取高质量基因组DNA提供了一种简单、经济高效且可重复的方法，适用于PCR、基因分型和测序应用。
