Pan'kov S V, Chechetkin V R, Somova O G, Antonova O V, Moiseeva O V, Prokopenko D V, Yurasov R A, Gryadunov D A, Chudinov A V
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov str., 32, 119991 Moscow, Russia.
J Biomol Struct Dyn. 2009 Oct;27(2):235-44. doi: 10.1080/07391102.2009.10507312.
Among various factors affecting operation of oligonucleotide microchips, the variations in concentration and in homogeneous distribution of immobilized probes over the cells are one of the most important. The labeling of immobilized probes ensures the complete current monitoring on the probe distribution and is reliable and convenient. Using hydrogel-based oligonucleotide microchips, the applicability of Cy3-labeled immobilized probes for quality control and signal normalization after hybridization with Cy5-labeled target DNA was investigated. This study showed that proper signal normalization should be different in thermodynamic conditions and in transient regime with hybridization far from saturation. This kinetic effect holds for both hydrogel-based and surface oligonucleotide microchips. Besides proving basic features, the technique was assessed on a sampling batch of 50 microchips developed for identifying mutations responsible for rifampicin and isoniazid resistance of Mycobacterium tuberculosis.
在影响寡核苷酸微芯片操作的各种因素中，固定化探针在细胞上的浓度变化和均匀分布是最重要的因素之一。固定化探针的标记可确保对探针分布进行完整的电流监测，且可靠便捷。使用基于水凝胶的寡核苷酸微芯片，研究了Cy3标记的固定化探针在与Cy5标记的靶DNA杂交后用于质量控制和信号归一化的适用性。该研究表明，在热力学条件和杂交远未饱和的瞬态状态下，适当的信号归一化应该有所不同。这种动力学效应适用于基于水凝胶的和表面寡核苷酸微芯片。除了证明基本特征外，该技术还在一批为鉴定导致结核分枝杆菌对利福平和异烟肼耐药的突变而开发的50个微芯片的样本上进行了评估。
