Institute of Theoretical and Experimental Biophysics, Russian Academy of Science, Pushchino 142290, Moscow Region, Russia.
Anal Biochem. 2010 Apr 1;399(1):125-31. doi: 10.1016/j.ab.2009.12.030. Epub 2009 Dec 28.
The efficiency of hybridization analysis with oligonucleotide microarrays depends heavily on the method of detection. Conventional methods based on labeling nucleic acids with fluorescent, chemiluminescent, enzyme, or radioactive reporters suffer from a number of serious drawbacks which demand development of new detection techniques. Here, we report two new approaches for detection of hybridization with oligonucleotide microarrays employing magnetic beads as active labels. In the first method streptavidin-coated magnetic beads are used to discover biotin-labeled DNA molecules hybridized with arrayed oligonucleotide probes. In the second method biotin-labeled DNA molecules are bound first to the surface of magnetic beads and then hybridized with arrayed complementary strands on bead-array contacts. Using a simple low-power microscope with a dark-field illumination and a pair of complementary primers as a model hybridization system we evaluated sensitivity, speed, and cost of the new detection method and compared its performance with the detection techniques employing enzyme and fluorescent labels. It was shown that the detection of microarray-hybridized DNA with magnetic beads combines low cost with high speed and enhanced assay sensitivity, opening a new way to routine hybridization assays which do not require precise measurements of DNA concentration.
寡核苷酸微阵列杂交分析的效率在很大程度上取决于检测方法。基于用荧光、化学发光、酶或放射性报告物标记核酸的传统方法存在许多严重的缺点,这需要开发新的检测技术。在这里,我们报告了两种使用磁性珠作为活性标记物检测寡核苷酸微阵列杂交的新方法。在第一种方法中,链霉亲和素包被的磁性珠用于发现与阵列寡核苷酸探针杂交的生物素标记的 DNA 分子。在第二种方法中,首先将生物素标记的 DNA 分子结合到磁性珠的表面,然后与珠阵列接触上的阵列互补链杂交。使用带有暗场照明的简单低功率显微镜和一对互补引物作为模型杂交系统,我们评估了新检测方法的灵敏度、速度和成本,并将其性能与使用酶和荧光标记的检测技术进行了比较。结果表明,使用磁性珠检测微阵列杂交 DNA 结合了低成本、高速和增强的检测灵敏度,为不需要精确测量 DNA 浓度的常规杂交分析开辟了新途径。