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On-chip optical detection system for allele-specific extension of 3'-LNA modified oligonucleotides
https://repo.qst.go.jp/records/68379
https://repo.qst.go.jp/records/683790c791a3b-a12a-4656-83ff-ba8bc7e04855
| Item type | 会議発表用資料 / Presentation(1) | |||||
|---|---|---|---|---|---|---|
| 公開日 | 2006-06-05 | |||||
| タイトル | ||||||
| タイトル | On-chip optical detection system for allele-specific extension of 3'-LNA modified oligonucleotides | |||||
| 言語 | ||||||
| 言語 | eng | |||||
| 資源タイプ | ||||||
| 資源タイプ識別子 | http://purl.org/coar/resource_type/c_c94f | |||||
| 資源タイプ | conference object | |||||
| アクセス権 | ||||||
| アクセス権 | metadata only access | |||||
| アクセス権URI | http://purl.org/coar/access_right/c_14cb | |||||
| 著者 |
Michikawa, Yuichi
× Michikawa, Yuichi× Fujimoto, Kentaro× Kinoshita, Kenji× Kawai, Seiko× Sugahara, Keisuke× Suga, Tomo× Ootsuka, Yoshimi× Fujiwara, Kazuhiko× Iwakawa, Mayumi× Imai, Takashi× 道川 祐市× 川井 聖子× 菅原 圭亮× 菅 智× 荘司 好美× 岩川 眞由美× 今井 高志 |
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| 抄録 | ||||||
| 内容記述タイプ | Abstract | |||||
| 内容記述 | Single nucleotide polymorphisms (SNPs) are useful as genetic association markers for various human diseases as well as for prediction of individual responses to therapeutic treatment such as drugs and ionizing radiation. For routine molecular biology research and bedside clinical diagnosis, readily available technologies are required to genotype limited numbers of SNPs that were selected in previous large scale association studies. To this end, easy and rapid protocols with inexpensive instruments running at reasonable cost are required for the technology to be widely adopted. \n In the present work, a novel optical detection system for on-chip allele-specific primer extension has been developed to conveniently genotype multiple SNPs. Optimization of the procedure was achieved by i) locked nucleic acid (LNA) modification of the 3'-end of immobilized oligonucleotide primer, ii) titration of magnesium concentration of the reaction mixture iii) utilization of optimum reaction temperature. Efficient primer extension without an annealing step using double-stranded template DNAs was demonstrated for LNA-modified oligonucleotides immobilized on an S-Bio PrimeSurface plastic base. This property provided simplification of experimental procedures and reduction of reaction time to as short as 10 minutes at a constant temperature of 65 oC. Incorporation of biotin-dUTP during primer extension, followed by binding of alkaline phosphatase-conjugated streptavidin, allowed optical detection of the typing results through precipitation of colored alkaline phosphatase substrate onto the surface of the plastic base. Oligonucleotide primer sets were designed to genotype three SNPs in the genes APEX1, TGFB1 and SOD2, previously investigated for association with radiation sensitivity. The simultaneous evaluation of these SNPs in 25 individuals has produced considerably reliable results. \n The experimental system developed in this study is not oriented towards high throughput analysis. Rather, limited numbers of SNPs are easily analyzed within a couple of hours. Dividing the surface of the plastic base into multiple areas can increase the number of individuals analyzed per chip without affecting experimental processes. In conclusion, all the benefits described above make this system applicable to routine molecular biology research and bedside clinical diagnosis. |
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| 会議概要（会議名, 開催地, 会期, 主催者等） | ||||||
| 内容記述タイプ | Other | |||||
| 内容記述 | HUGO's 11th Human Genome Meeting （HGM2006) | |||||
| 発表年月日 | ||||||
| 日付 | 2006-06-03 | |||||
| 日付タイプ | Issued | |||||
