A high-throughput, high-quality plant genomic DNA extraction protocol

文献类型： 外文期刊

第一作者： Li, H.

作者： Li, H.;Li, J.;Li, H.;Li, J.;Cong, X. H.;Duan, Y. B.;Li, L.;Wei, P. C.;Lu, X. Z.;Yang, J. B.;Duan, Y. B.

作者机构：

关键词： Genomic DNA extraction;Liquid nitrogen-free protocol;High-throughput protocol;Phenol-chloroform-free protocol;High-quality protocol;Real-time PCR

期刊名称：GENETICS AND MOLECULAR RESEARCH （ 影响因子：0.764； 五年影响因子：0.912 ）

ISSN： 1676-5680

年卷期： 2013 年 12 卷 4 期

页码：

收录情况： SCI

摘要： 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 mu g 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/mu L). 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.

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