Sorokin N V, Chechetkin V R, Pan'kov S V, Somova O G, Livshits M A, Donnikov M Y, Turygin A Y, Barsky V E, Zasedatelev A S
Engelhardt Institute of Molecular Biology of Russian Academy of Sciences, Vavilov str., 32, Moscow, Russia 119991.
J Biomol Struct Dyn. 2006 Aug;24(1):57-66. doi: 10.1080/07391102.2006.10507099.
The optimal design of oligonucleotide microchips and efficient discrimination between perfect and mismatch duplexes strongly depend on the external transport of target DNA to the cells with immobilized probes as well as on respective association and dissociation rates at the duplex formation. In this paper we present the relevant theory for hybridization of DNA fragments with oligonucleotide probes immobilized in the cells on flat substrate. With minor modifications, our theory also is applicable to reaction-diffusion hybridization kinetics for the probes immobilized on the surface of microbeads immersed in hybridization solution. The main theoretical predictions are verified with control experiments. Besides that, we compared the characteristics of the surface and gel-based oligonucleotide microchips. The comparison was performed for the chips printed with the same pin robot, for the signals measured with the same devices and processed by the same technique, and for the same hybridization conditions. The sets of probe oligonucleotides and the concentrations of probes in respective solutions used for immobilization on each platform were identical as well. We found that, despite the slower hybridization kinetics, the fluorescence signals and mutation discrimination efficiency appeared to be higher for the gel-based microchips with respect to their surface counterparts even for the relatively short hybridization time about 0.5-1 hour. Both the divergence between signals for perfects and the difference in mutation discrimination efficiency for the counterpart platforms rapidly grow with incubation time. In particular, for hybridization during 3 h the signals for gel-based microchips surpassed their surface counterparts in 5-20 times, while the ratios of signals for perfect-mismatch pairs for gel microchips exceeded the corresponding ratios for surface microchips in 2-4 times. These effects may be attributed to the better immobilization efficiency and to the higher thermodynamic association constants for duplex formation within gel pads.
寡核苷酸微芯片的优化设计以及完美双链体与错配双链体之间的有效区分，在很大程度上取决于目标DNA向固定有探针的细胞的外部传输，以及双链体形成时各自的结合和解离速率。在本文中，我们提出了DNA片段与固定在平面基底细胞中的寡核苷酸探针杂交的相关理论。经过微小修改，我们的理论也适用于固定在浸入杂交溶液中的微珠表面的探针的反应扩散杂交动力学。主要理论预测通过对照实验得到验证。除此之外，我们比较了基于表面和凝胶的寡核苷酸微芯片的特性。比较是针对用相同点样机器人打印芯片、用相同设备测量并通过相同技术处理信号，以及在相同杂交条件下进行的。用于固定在每个平台上的探针寡核苷酸组和各自溶液中探针的浓度也相同。我们发现，尽管杂交动力学较慢，但即使对于约0.5 - 1小时的相对较短杂交时间，基于凝胶的微芯片相对于其表面对应物，荧光信号和突变区分效率似乎更高。完美双链体信号之间的差异以及对应平台的突变区分效率差异都随孵育时间迅速增加。特别是，在3小时杂交过程中，基于凝胶的微芯片的信号比其表面对应物高出5 - 20倍，而凝胶微芯片的完美 - 错配双链体信号比值比表面微芯片的相应比值高出2 - 4倍。这些效应可能归因于更好的固定效率以及凝胶垫内双链体形成的更高热力学结合常数。
