Bavykin S G, Akowski J P, Zakhariev V M, Barsky V E, Perov A N, Mirzabekov A D
BioChip Technology Center, Argonne National Laboratory, Argonne, Illinois 60439, USA.
Appl Environ Microbiol. 2001 Feb;67(2):922-8. doi: 10.1128/AEM.67.2.922-928.2001.
We have developed a three-component system for microbial identification that consists of (i) a universal syringe-operated silica minicolumn for successive DNA and RNA isolation, fractionation, fragmentation, fluorescent labeling, and removal of excess free label and short oligonucleotides; (ii) microarrays of immobilized oligonucleotide probes for 16S rRNA identification; and (iii) a portable battery-powered device for imaging the hybridization of fluorescently labeled RNA fragments with the arrays. The minicolumn combines a guanidine thiocyanate method of nucleic acid isolation with a newly developed hydroxyl radical-based technique for DNA and RNA labeling and fragmentation. DNA and RNA can also be fractionated through differential binding of double- and single-stranded forms of nucleic acids to the silica. The procedure involves sequential washing of the column with different solutions. No vacuum filtration steps, phenol extraction, or centrifugation is required. After hybridization, the overall fluorescence pattern is captured as a digital image or as a Polaroid photo. This three-component system was used to discriminate Escherichia coli, Bacillus subtilis, Bacillus thuringiensis, and human HL60 cells. The procedure is rapid: beginning with whole cells, it takes approximately 25 min to obtain labeled DNA and RNA samples and an additional 25 min to hybridize and acquire the microarray image using a stationary image analysis system or the portable imager.
我们开发了一种用于微生物鉴定的三组分系统，该系统包括：（i）一个通用的注射器操作硅胶微柱，用于连续进行DNA和RNA的分离、分级分离、片段化、荧光标记以及去除过量的游离标记和短寡核苷酸；（ii）用于16S rRNA鉴定的固定化寡核苷酸探针微阵列；（iii）一个便携式电池供电设备，用于对荧光标记的RNA片段与阵列的杂交进行成像。该微柱将硫氰酸胍核酸分离方法与新开发的基于羟基自由基的DNA和RNA标记及片段化技术相结合。DNA和RNA也可以通过核酸双链和单链形式与硅胶的差异结合进行分级分离。该过程包括用不同溶液依次洗涤柱子。无需真空过滤步骤、苯酚提取或离心。杂交后，整体荧光模式以数字图像或宝丽来照片的形式捕获。这个三组分系统用于区分大肠杆菌、枯草芽孢杆菌、苏云金芽孢杆菌和人HL60细胞。该过程很快：从全细胞开始，获得标记的DNA和RNA样品大约需要25分钟，使用固定图像分析系统或便携式成像仪进行杂交并获取微阵列图像还需要额外25分钟。
