Agbagwa I O, Datta S, Patil P G, Singh P, Nadarajan N
Crop Improvement Division, Indian Institute of Pulses Research, Kanpur, India.
Genet Mol Res. 2012 Dec 19;11(4):4632-9. doi: 10.4238/2012.September.14.1.
Current DNA extraction protocols, which require liquid nitrogen, lyophilization and considerable infrastructure in terms of instrumentation, often impede the application of biotechnological tools in less researched crops in laboratories in developing countries. We modified and optimized the existing CTAB method for plant genomic DNA extraction by avoiding liquid nitrogen usage and lyophilization. DNA was extracted directly from freshly harvested leaves ground in pre-heated CTAB buffer. Chloroform:isoamyl alcohol (24:1) and RNase treatments followed by single-purification step decontaminated the samples thereby paving way for selective extraction of DNA. High molecular weight DNA yield in the range of 328 to 4776 ng/μL with an average of 1459 ng/μL was obtained from 45 samples of cultivated and wild Cajanus species. With an absorbance ratio at 260 to 280 nm, a range of 1.66 to 2.20, and a mean of 1.85, very low levels of protein and polysaccharide contamination were recorded. Forty samples can be extracted daily at a cost between 1.8 and US$2.0 per plant sample. This modified method is suitable for most plants especially members of the Leguminosae. Apart from Cajanus, it has been extensively applied in DNA extraction from Cicer and Vigna species.
当前的DNA提取方案需要液氮、冻干以及大量的仪器设备基础设施，这常常阻碍了生物技术工具在发展中国家实验室中对研究较少的作物的应用。我们通过避免使用液氮和冻干对现有的用于植物基因组DNA提取的CTAB方法进行了改进和优化。DNA直接从在预热的CTAB缓冲液中研磨的新鲜收获叶片中提取。氯仿：异戊醇（24:1）和核糖核酸酶处理，随后进行单步纯化步骤对样品进行了净化，从而为DNA的选择性提取铺平了道路。从45个栽培和野生木豆属物种的样品中获得了高分子量DNA，产量在328至4776 ng/μL之间，平均为1459 ng/μL。在260至280 nm的吸光度比值范围为1.66至2.20，平均值为1.85，记录到蛋白质和多糖污染水平非常低。每天可以提取40个样品，每个植物样品的成本在1.8美元至2.0美元之间。这种改进的方法适用于大多数植物，尤其是豆科植物成员。除了木豆属，它还被广泛应用于从鹰嘴豆属和豇豆属物种中提取DNA。
