羧甲基壳聚糖磁性纳米复合物高效分离基因组DNA

Extraction of genomic DNA by carboxymethylated chitosan-Fe_3O_4 nanocomposites

分别以常量的大肠杆菌和微量的嗜酸氧化亚铁硫杆菌(Acidithiobacillus ferrooxidans)为实验对象,研究羧甲基壳聚糖磁性纳米复合物载体对基因组DNA的分离纯化,对分离的大肠杆菌基因组DNA直接采用琼脂糖凝胶电泳和紫外分光光度计分析,分离的A.ferrooxidans基因组DNA则直接作为硫化物质体醌氧化还原酶基因片断的PCR扩增模板。研究结果表明:采用磁性纳米复合物法,其制备DNA的时间是传统方法的1/3,具有快速、简单的优点,在微量样品DNA提取中,由于其富集提取DNA,并提高了PCR的灵敏性功能,比传统方法优越,可用于食品卫生致病微生物的检测、法医鉴定以及极难培养的微生物的菌种鉴定。

The carboxymethylated chitosan-Fe3O4 nanocomposites were applied to extract genomic DNA from Escherichia coli and traces of Acidithiobacillus ferrooxidans strains, respectively. The quality of isolated DNA from E. coli was analyzed directly by agarose gel electrophoresis and UV spectrometry, and DNA isolated from A. ferrooxidans strains was directly used as DNA template for PCR amplification of gene fragment of sulfide quinone oxidoreductase, and the results obtained with magnetic nanocomposites were compared with those of conventional phenol-chloroform extraction. The results show that the magnetic nanocomposites are suitable for fast extraction of genomic DNA from samples without involving the use of hazardous agents, simplifying extremely the whole procedure for DNA preparation and enhancing sensitivity of PCR amplification. It can be used to detect pathogenic bacteria in food hygiene, judge forensic medicine and identify some microorganisms that are difficult to culture.