Santosa D A
Department of Soil Science, Faculty of Agriculture, Bogor Agricultural University, and Indonesian Center for Biodiversity and Biotechnology (ICBB), R.E. Martadinata 8, Bogor 16162, Indonesia.
Mol Biotechnol. 2001 Jan;17(1):59-64. doi: 10.1385/MB:17:1:59.
A rapid method for the extraction and purification of DNA from environmental samples for molecular cloning applications was developed. The indigenous cells from plant debris, organic materials, sediments, and soils were lysed directly by using DAS-IZ solution and the nucleic acids were precipitated with isopropanol. A simple purification step using DAS-IIZ solution without binding matrix produced highly pure, colorless and undegraded DNA with molecular weight of more than 20 kb. The superiority of this method was tested for wide applications in molecular cloning, i.e., construction of genomic library by using Lambda DASHII Vector and GigapackIII XL, plasmid library, cloning of gene encoding protease, and molecular microbial diversity analysis. An additional advantage of this method is that only 0.1 g of sample is required, if analysis of many samples in short time should be done. To extract large amounts of environmental DNA for molecular cloning lasts only 30 min and to purify it less than 1 h.
开发了一种从环境样品中提取和纯化用于分子克隆应用的DNA的快速方法。使用DAS-IZ溶液直接裂解来自植物碎片、有机材料、沉积物和土壤中的原生细胞，并用异丙醇沉淀核酸。使用不含结合基质的DAS-IIZ溶液进行的简单纯化步骤产生了分子量超过20 kb的高纯度、无色且未降解的DNA。该方法的优越性在分子克隆的广泛应用中得到了测试，即使用Lambda DASHII载体和GigapackIII XL构建基因组文库、质粒文库、编码蛋白酶的基因克隆以及分子微生物多样性分析。该方法的另一个优点是，如果要在短时间内分析许多样品，仅需0.1 g样品。提取大量用于分子克隆的环境DNA仅需30分钟，纯化则不到1小时。
