Li H, Li J, Cong X H, Duan Y B, Li L, Wei P C, Lu X Z, Yang J B
Key Laboratory of Ion Beam Bioengineering, Institute of Technical Biology and Agriculture Engineering, Chinese Academy of Sciences, Hefei, China.
Genet Mol Res. 2013 Oct 15;12(4):4526-39. doi: 10.4238/2013.October.15.1.
The isolation of high-quality genomic DNA (gDNA) is a crucial technique in plant molecular biology. The quality of gDNA determines the reliability of real-time polymerase chain reaction (PCR) analysis. In this paper, we reported a high-quality gDNA extraction protocol optimized for real-time PCR in a variety of plant species. Performed in a 96-well block, our protocol provides high throughput. Without the need for phenol-chloroform and liquid nitrogen or dry ice, our protocol is safer and more cost-efficient than traditional DNA extraction methods. The method takes 10 mg leaf tissue to yield 5-10 µg high-quality gDNA. Spectral measurement and electrophoresis were used to demonstrate gDNA purity. The extracted DNA was qualified in a restriction enzyme digestion assay and conventional PCR. The real-time PCR amplification was sufficiently sensitive to detect gDNA at very low concentrations (3 pg/µL). The standard curve of gDNA dilutions from our phenol-chloroform-free protocol showed better linearity (R(2) = 0.9967) than the phenol-chloroform protocol (R(2) = 0.9876). The results indicate that the gDNA was of high quality and fit for real-time PCR. This safe, high-throughput plant gDNA extraction protocol could be used to isolate high-quality gDNA for real-time PCR and other downstream molecular applications.
高质量基因组DNA（gDNA）的分离是植物分子生物学中的一项关键技术。gDNA的质量决定了实时聚合酶链反应（PCR）分析的可靠性。在本文中，我们报道了一种针对多种植物物种的实时PCR优化的高质量gDNA提取方案。该方案在96孔板中进行，具有高通量。无需酚-氯仿以及液氮或干冰，我们的方案比传统DNA提取方法更安全且成本效益更高。该方法使用10毫克叶片组织可产生5-10微克高质量gDNA。通过光谱测量和电泳来证明gDNA的纯度。提取的DNA在限制性内切酶消化试验和常规PCR中合格。实时PCR扩增足够灵敏，能够检测到极低浓度（3 pg/µL）的gDNA。我们的无酚-氯仿方案的gDNA稀释标准曲线显示出比酚-氯仿方案（R(2) = 0.9876）更好的线性（R(2) = 0.9967）。结果表明gDNA质量高且适合实时PCR。这种安全、高通量的植物gDNA提取方案可用于分离高质量gDNA，用于实时PCR和其他下游分子应用。
