Fotin A V, Drobyshev A L, Proudnikov D Y, Perov A N, Mirzabekov A D
Joint Human Genome Program: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 117984 Moscow, Russia.
Nucleic Acids Res. 1998 Mar 15;26(6):1515-21. doi: 10.1093/nar/26.6.1515.
A microchip method has been developed for massive and parallel thermodynamic analyses of DNA duplexes. Fluorescently labeled oligonucleotides were hybridized with oligonucleotides immobilized in the 100 x 100 x 20 mum gel pads of the microchips. The equilibrium melting curves for all microchip duplexes were measured in real time in parallel for all microchip duplexes. Thermodynamic data for perfect and mismatched duplexes that were obtained using the microchip method directly correlated with data obtained in solution. Fluorescent labels or longer linkers between the gel and the oligonucleotides appeared to have no significant effect on duplex stability. Extending the immobilized oligonucleotides with a four-base mixture from the 3'-end or one or two universal bases (5-nitroindole) from the 3'- and/or 5'-end increased the stabilities of their duplexes. These extensions were applied to increase the stabilities of the duplexes formed with short oligonucleotides in microchips, to significantly lessen the differences in melting curves of the AT- and GC-rich duplexes, and to improve discrimination of perfect duplexes from those containing poorly recognized terminal mismatches. This study explored a way to increase the efficiency of sequencing by hybridization on oligonucleotide microchips.
已开发出一种用于对DNA双链体进行大规模并行热力学分析的微芯片方法。将荧光标记的寡核苷酸与固定在微芯片100×100×20μm凝胶垫中的寡核苷酸杂交。对所有微芯片双链体实时并行测量其平衡解链曲线。使用微芯片方法获得的完美双链体和错配双链体的热力学数据与在溶液中获得的数据直接相关。凝胶与寡核苷酸之间的荧光标记或较长连接子似乎对双链体稳定性没有显著影响。从3'-末端用四碱基混合物或从3'-和/或5'-末端用一个或两个通用碱基（5-硝基吲哚）延伸固定化寡核苷酸可提高其双链体的稳定性。这些延伸用于提高微芯片中与短寡核苷酸形成的双链体的稳定性，显著减小富含AT和富含GC的双链体解链曲线的差异，并改善对完美双链体与含有识别不佳的末端错配的双链体的区分。本研究探索了一种提高寡核苷酸微芯片上杂交测序效率的方法。
