Abstract:

Objective Traditional plant genomic DNA extraction methods (such as the CTAB method and SDS lysis) are often labor-intensive, time-consuming, and reliant on toxic organic reagents, making them unsuitable for the growing demand of high-throughput molecular detection. This study aims to develop a rapid, safe, and high-throughput method for plant DNA extraction to improve the efficiency of large-scale sample processing. Method We proposed a fast DNA extraction method based on 96-deep-well plates, referred to as the DDEB method (directPCR DNA extraction buffer). The DDEB extraction buffer comprised 0.2 mol/L NaOH, 0.01% SDS, 50 mmol/L NaCl, 0.1 mmol/L EDTA-2Na, 0.15 g/L gelatin, and 0.005% antifoaming agent A. During extraction, samples were mixed with the buffer and subjected to oscillatory grinding, followed by brief centrifugation or static settling to obtain crude DNA extracts suitable for direct PCR amplification. Result The DDEB method enabled the extraction of DNA from hundreds of plant samples within 5 min. The resulting crude DNA, after 5‒20-fold dilution, can be directly used as a PCR template. Using young leaves of oilseed rape (Brassica napus) and rice (Oryza sativa) as material, the target fragments up to 2 000 bp were successfully amplified, with clear and specific bands. The method performed well in molecular marker analysis, yielding accurate and reproducible genotyping results. It was further applied to construct a partial linkage map of oilseed rape. Conclusion This study establishes a “rapid-safe-high-throughput” strategy for plant genomic DNA extraction. This approach significantly simplifies the traditional DNA extraction process while maintaining reliable PCR amplification and genotyping accuracy. It markedly improves operational efficiency and is especially suitable for large-scale genotyping of plant samples, offering a cost-effective and efficient solution for molecular breeding and germplasm research.

Key words: DNA extraction, CTAB method, direct PCR, PCR amplification, molecular marker, Brassica napus, Oryza sativa, high-throughput