一种高通量快速提取植物基因组DNA的方法

doi:10.13560/j.cnki.biotech.bull.1985.2025-0662

摘要/Abstract

摘要：

目的传统植物基因组DNA提取方法（如CTAB法、SDS裂解法等）存在操作繁琐、耗时长、使用有毒有机试剂等问题，难以满足当前高通量分子检测的实际需求。旨在开发一种快速、安全、适用于大规模样本处理的植物DNA提取方法，以提高分子检测效率。方法提出一种基于96深孔板的快速DNA提取方法——DDEB（directPCR DNA extraction buffer）法。该方法采用DDEB提取液，主要成分包括0.2 mol/L NaOH、0.01% SDS、50 mmol/L NaCl、0.1 mmol/L EDTA-2Na、0.15 g/L明胶及0.005%消泡剂A等组分。提取时将样品与提取液混合后通过振荡研磨，继而瞬时离心或静置沉降，即可获得可用于PCR扩增的DNA粗提液。结果该方法在5 min内完成数百份植物样品的DNA提取，所得DNA稀释5‒20倍即可直接用于PCR模板使用。以油菜（Brassica napus）和水稻（Oryza sativa）幼叶为材料提取DNA，成功扩增出2 000 bp以内的目的片段，扩增条带清晰、特异性良好。应用该方法进行分子标记分析，分型结果准确，重复性高，并成功用于构建油菜的局部遗传连锁图谱。结论建立一种“快速‒安全‒高通量”的植物DNA提取策略，简化了传统DNA提取流程，在保证扩增效果和分型准确性的基础上，大幅提升了实验效率，尤其适用于大规模植物样品的基因型检测，为分子育种和种质资源研究提供了高效、低成本的技术解决方案。

关键词: DNA提取, CTAB法, 直扩法, PCR扩增, 分子标记, 油菜, 水稻, 高通量

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

杨洋, 刘慧敏, 林俐, 王幼平, 吴健. 一种高通量快速提取植物基因组DNA的方法[J]. 生物技术通报, 2025, 41(10): 156-163.

YANG Yang, LIU Hui-min, LIN Li, WANG You-ping, WU Jian. A High-throughput and Rapid Method for Plant Genomic DNA Extraction[J]. Biotechnology Bulletin, 2025, 41(10): 156-163.

图/表 6

图1 DDEB法快速制备植物基因组DNA的操作步骤示意图

Fig. 1 Schematic diagram of the operational steps of DDEB method for the preparation of plant genomic DNA

表1 本研究中所用的引物序列

Table 1 Primer sequences used in this study

引物名称 Primer name	正向引物序列 Forward primer sequence （5′‒3′）	反向引物序列 Reverse primer sequence （5′‒3′）	产物大小 Amplicon size （bp）
BnaC06.IDA	CAAACCAGATTCCCATTTCC	GGAATGGGAACACCTTTGGG	514
BnaA07.IDA	AAGCGAAAAGATAAGCTTGC	CGGATGATGTGTGATGCTGGA	974
ProBnaC06.IDA	GGGTGAACCCAAGAATAAC	TCGCCGCCAGAAACAGAACC	1 215
ProBnaA07.IDA	CTTGGGGCAACCCTAATCTG	CACGGAGCCATTTGGTAAT	1 634
ProBnaC04.IDA	CCACCACAAACGAGAAGACG	CACGGAGCCATTTGGTAATG	1 803
OsActin	GGTGTCATGGTCGGAATGGG	GTCCAGGGCGATGTAGGAAA	792
InDel205	TTAAGCCCAAAACAAATTCAAATGGACTAAAA	CAAAATTTGGTTGCATAACCCTATTTCTCTTT	127
InDel348	GAAAATAAAATTGGGAGTGGGAGGGAAA	TTTTCTCTAACGTCTCCACACGAAGATTAT	130
InDel299	GAAACTAACTAATTTCACTTTCTCGCTCATCT	CTTTGATACTGTTGAACTAAACCGACTTTTCT	101
InDel322	CACCCACAAACCAAAAACTAAAATTATACGAA	GCTGTAGTCTCGGTATCTCAGATTGTG	116
InDel204	GAAGAAGCTTCGAATTTGACGGTGAC	GTCATTATTTTATGTATTTCATCCCGGGCG	108
InDel295	AAAGCTGGCACGTCACAATATCAAT	CGAGCTATCTTTTGTTTTGGTGATATATCCTT	130
InDel319	AGTTTTGAGTCGTATCTGAAATAAACAAAAGA	AGCACTACTTGTCGGAAAATAAAATAACATGA	130
InDel285	TAAAAATCATTATGCAGACCGTAACTGGTAAT	ATGATGAAAATCTAAAGTGTTTTCCGTCAAAG	106
InDel620	AGGAAAACCCCCAAAAGTAAATTTAAAAACAT	TATTGATTTTCCTCCACTGATTTGGTTTTCTA	104
InDel599	CCATTGTAAACATCCTACGTATTGATTACTGA	CAAGCTTGTGAAAAGAAAGAAGAAGAAAATGT	116
InDel605	GACTGATACCCACATGTATTAGAAGCAATGAC	GACTTTTACCCGGAACTGTTTACTTGAA	119

图2 DDEB法和CTAB法制备的油菜基因组DNA在不同稀释浓度下的PCR扩增效果比较A‒E扩增大小分别为514 bp（A）、974 bp（B）、1 215 bp（C）、1 634 bp（D）、1 803 bp（E）；M：DNA marker

Fig. 2 Comparative PCR amplification efficiency of rapeseed genomic DNA extracted by DDEB and CTAB methods across varying dilution gradientsA‒E the amplified fragment sizes are 514 bp (A), 974 bp (B), 1 215 bp (C), 1 634 bp (D), and 1 803 bp (E), respectively; M: DNA marker

图3 DDEB法和CTAB法制备的水稻基因组DNA在不同稀释浓度下的PCR扩增效果比较

Fig. 3 Comparative PCR amplification efficiency of rice genomic DNA extracted by DDEB and CTAB methods across varying dilution gradients

图4 DDEB法和CTAB法制备的油菜基因组DNA在InDel分子标记检测中的效果比较P1：皖油29号；P2：SWU66；F1：‘皖油29’为母本，‘SWU66’为父本的杂交一代

Fig. 4 Comparative evaluation of DDEB and CTAB methods for rapeseed genomic DNA preparation in InDel marker analysisP1: Wanyou 29; P2: SWU66; F1: the hybrid of 'Wanyou 29' (female parent)×'SWU66' (male parent)

图5 基于DDEB法提取的油菜基因组DNA构建的局部遗传连锁图谱分析A：F₂群体InDel204标记基因型检测结果（聚丙烯酰胺凝胶电泳图）；B：局部遗传连锁图谱，图示从上至下依次为标记名称、遗传距离（cM）及对应物理位置（Mb）

Fig. 5 Analysis of partial genetic linkage map constructed using rapeseed genomic DNA extracted by the DDEB methodA: Genotyping results of InDel204 marker in F₂ population (polyacrylamide gel electrophoresis image). B: Partial genetic linkage map showing (from top to bottom): marker names, genetic distances (cM), and corresponding physical positions (Mb)

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