| Budget Amount *help |
¥17,360,000 (Direct Cost: ¥15,500,000、Indirect Cost: ¥1,860,000)
Fiscal Year 2007: ¥8,060,000 (Direct Cost: ¥6,200,000、Indirect Cost: ¥1,860,000)
Fiscal Year 2006: ¥9,300,000 (Direct Cost: ¥9,300,000)
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| Research Abstract |
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
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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
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