Parallel thermodynamic analysis of duplexes on oligodeoxyribonucleotide microchips
Parallel thermodynamic analysis of duplexes on oligodeoxyribonucleotide microchips
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DOI：
10.1093/nar/26.6.1515
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发表时间：
1998-03-15
影响因子：
14.9
通讯作者：
MIrzabekov, AD
中科院分区：
文献类型：
--
作者：
Fotin, AV;Drobyshev, AL;MIrzabekov, AD
发展了一种用于DNA双链体的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的100 × 100 × 20 μ m凝胶垫中的寡核苷酸杂交。所有微芯片双链体的平衡解链曲线都是在真实的时间内对所有微芯片双链体平行测量的。使用微芯片方法获得的完美和错配双链体的热力学数据与溶液中获得的数据直接相关。凝胶和寡核苷酸之间的荧光标记或更长的接头似乎对双链体稳定性没有显著影响。从3 ′-端用四碱基混合物或从3 ′-和/或5 ′-端用一个或两个通用碱基（5-硝基吲哚）延伸固定的寡核苷酸增加了它们的双链体的稳定性。这些.应用延伸以增加微芯片中由短寡核苷酸形成的双链体的稳定性，以显著减少AT-和GC-富集双链体的解链曲线的差异，并改善完美双链体与含有识别不良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芯片杂交测序效率的方法。
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 mu m 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.