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Research paper : Development of novel chemical reagents for reliable genetic analyses (Y. Komatsu et al.)−2−Synthesiology - English edition Vol.4 No.1 (2011) are also used to conjugate with fluorophores or drugs to the DNA (Fig. 2b). Under such circumstances, we determined that the “linkers” played an extremely important role among the “synthetic DNAs”. Similarly, we thought that the reactivity of the reagents to “label” genes recovered from samples not only would affect the sensitivity in detecting minute amounts of genes (Fig. 2c), but also may lead to the development of nucleic acid drugs. Therefore, we focused on the issues in conventional linkers and labeling reagents, and aimed to develop a new type of reagent with higher performance. After describing the development and the product realization of the linker used for probe modification, we shall present the development of the labeling reagent.4 Development and results4.1 Development of the amino linker4.1.1 Goal of the developmentThe DNA used as a probe is synthesized sequentially by coupling the four monomer building blocks - adenine, guanine, cytosine, and thymine, according to sequence information. At the same time, the linker needed for the immobilization of the DNA to solid surface is incorporated to the termini of synthesized DNAs by using the specific “linker reagent” at the final step of the synthesis. Although there are several types of linkers possessing the different functional groups, the amino linker with primary amino group is used most frequently for the chemical modification of DNA because of the chemical stability and the easy handling.DNAs modified with the amino group (amino-modified DNA) are generally conducted by specialized DNA synthesis companies. In one process of this synthesis, defective DNAs failing in the linker incorporation must be separated from amino-modified ones. However, since the presence of an amino group presents only a slight chemical difference, it is difficult to conduct this separation in a short time period. On the other hand, due to the recent demand for comprehensive genetic analysis of whole genomes, it has become necessary to prepare several hundreds to several thousands of amino-modified DNAs in parallel. However, we imagined that the DNA synthesis companies were facing trouble in their synthesis and purification processes due to the use of the conventional linker. Also recently, there is an increased potential for oligonucleotide drugs such as aptamers and siRNA. In order to increase their functions in vivo, various functional compounds must be conjugated with oligonucleotides through the linker with high yields, and we thought there would be a high demand for increasing the reactivity of the amino group as well as simplifying the purification process (Fig. 2B, Table 1).Therefore, we decided to develop a new amino linker that enabled both the high-purity high-throughput purification of amino-modified DNA (or RNA) as well as the high chemical modification efficiency, so it could be utilized in the recent comprehensive genetic analysis and the nucleic acid drugs.4.1.2 Development of the first-generation amino linkerThe conventional amino linker has a simple structure where a primary amino group is linked to the terminal of the straight carbon chain. We first developed a series of amino linkers which consist of a single aromatic molecule (Fig. 3, first-generation amino linker). We expected that the reaction efficiency would be enhanced by hydrophobic interaction between the aromatic residue and the target molecule. Also, we expected that the separation using the reverse-phase column chromatography would become easy because of the increased hydrophobic property of the amino-modified DNA molecule.Several types of amino linker reagents with different distances between the aromatic and amino groups were synthesized (Fig. 3; L1, L2), and the chemical properties of the amino-modified DNA were examined. The first-generation amino linkers dramatically improved both efficiencies in the coupling to the primary amine and the purification compared to the conventional linker (Table 1)[3], and we applied for the patent jointly with a collaborating company for the first-generation amino linker in 2004. Elementaltechnology1LabelingSyntheticDNANewtechnologyAddition of higher functionImprovement of geneticanalysis precisionGenetic analysis BGenetic analysis AElementaltechnology2NewtechnologyFig. 1 Technological configuration of the genetic analysis systemThe conventional technologies such as synthetic DNA (RNA) and labeling are used commonly in several genetic analysis systems, and also are related to pharmaceuticals.Table 1 Advantages and disadvantages of amino linkers• Cost• Performance• Stability• Cost• Performance• Purification• ReactivityDisadvantages• Reactivity (> Conventional)• Purification (≧ Conventional)• Reactivity (> Conventional)• Purification (≧ Conventional)• Stability• Cost• PerformanceAdvantagesSecond-generationFirst-generationConventional

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