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

在这项研究中，我们正在改进一种产生DNA芯片的方法，这种方法带有“载体上的探针”，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，每个寡核苷酸都是以多孔玻璃为固相载体合成的，可以作为互补靶序列的固定化探针。首先，我们利用一种新型疏水连接剂和一种新型非水试剂对DNA探针在多孔玻璃树脂上的合成进行了优化。我们还提出了一种新的策略，称为“受保护的DNA探针(PDP)方法”，在这种方法中，适当保护的碱基可以高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的受控多孔玻璃上高效地合成dna探针，从而避免了dna pr…的耗时分离过程。更多的出窍。用固定在玻璃板上的PPP成功地识别了SNPs，这表明它具有潜在的应用价值。此外，我们还成功地发展了一种新的SNP分析方法，在平板上使用化学或光化学连接技术，具有很高的偶联效率。这些方法都表现出很高的匹配/不匹配识别能力。因此，这些技术在实际应用中显著提高了探针载体DNA芯片系统的碱基分辨能力。我们还试图开发用于基因表达分析的探针载体DNA微阵列。该多孔玻璃载体为120聚体长的寡核苷酸合成提供了良好的结果。产率几乎是使用交联型聚苯乙烯的20倍，交联型聚苯乙烯是合成寡核苷酸最受欢迎的载体。因此，通过使用不同长度的探针载体，可以开发出一种既能同时分析SNPs又能分析基因表达的经济仪器。较少

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