Improvement of DNA chip system with probe-on-carriers for practical uses

在这项研究中，我们正在改进一种方法，以“载体上的探针”生成DNA芯片，固定在固相上的寡核苷酸探针，用于实际应用。在该DNA芯片中，每个寡核苷酸作为固相载体在多孔玻璃上合成，并可作为互补靶序列的固定化探针。因此，高质量的DNA芯片可以制造容易和经济。首先，我们利用一种新的疏水连接剂和一种新的非水试剂来优化多孔玻璃树脂上DNA探针的合成。我们还提出了一种新的策略，称为“保护DNA探针（PDP）方法”，其中适当保护的碱基可以高度选择性地与互补碱基结合，即使不去除它们的碱基保护基团。这种PDP策略可以保证在高纯度可控多孔玻璃上高效合成DNA探针，从而消除了分离DNA探针的耗时过程。利用固定在玻璃板上的pdp成功地识别了snp，这表明了它的潜在用途。此外，我们成功地开发了一种新的方法，利用高耦合效率的化学或光化学结扎技术在板上进行SNP分析。结果表明，该方法具有较高的匹配/错配判别能力。因此，这些技术在实际应用中显著提高了探针载体DNA芯片系统的碱基识别能力。我们还尝试开发带有探针载体的DNA微阵列，用于基因表达分析。多孔玻璃支架为120摩尔长的寡核苷酸合成提供了极好的结果。产率几乎是使用交联聚苯乙烯的20倍，交联聚苯乙烯是合成寡核苷酸最常用的载体。因此，通过使用不同长度的探针-载体探针，可以开发出一种既能分析snp又能分析基因表达的经济装置。少

In this study, we are improving a method to generate DNA chips with "probe-on-carriers", immobilized oligonucleotide probes on the solid phase, for practical uses. In this DNA chip, each oligonucleotide was synthesized on porous glass as a solid phase-carrier, and can be used as an immobilized probe for the complementary target sequence. Therefore, high-quality DNA chips can be fabricated easily and economically.First, we optimized synthesis of DNA probes on porous glass resins by using a new hydrophobic linker and a new non-aqueous reagent for the deprotection process. We also proposed a new strategy called "Protected DNA Probes (PDP) method" in which appropriately protected bases can bind highly selectively to the complementary bases even without removal of their base protecting groups. This PDP strategy could guarantee highly efficient synthesis of DNA probes on controlled porous glass with high purity and thereby could eliminate the time-consuming procedures for isolation of DNA pr … More obes. SNPs were successfully recognized each other by using PDPs immobilized on glass plates, suggesting its potential usefulness. Moreover, we succeeded to develop new method for the SNP analysis by using a chemical or photochemical ligation technique on plates with high coupling efficiency. These methods showed markedly high match/mismatch discrimination ability. Therefore, these technologies resulted in significant improvement of the base discrimination ability in DNA chip system with probe-on-carriers for practical uses.We also tried to develop DNA microarrays with probe-on-carriers for gene expression analysis. The porous glass supports gave an excellent result for a 120 mer-long oligonucleotide synthesis. The yield was almost 20 times as much as by using cross-linked polystyrene, the most popular support for oligonucleotide synthesis. Therefore, by using probe-on-carriers with different probe lengths, an economical device, which could analyze not only SNPs but also gene expression at one time, could be developed. Less