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Solid-phase multiple displacement amplification for multi-locus genotyping of single chromosome molecules
https://repo.qst.go.jp/records/69116
https://repo.qst.go.jp/records/69116e64fa729-ac0c-4f0b-a137-01c2fa289c96
| Item type | 会議発表用資料 / Presentation(1) | |||||
|---|---|---|---|---|---|---|
| 公開日 | 2007-10-01 | |||||
| タイトル | ||||||
| タイトル | Solid-phase multiple displacement amplification for multi-locus genotyping of single chromosome molecules | |||||
| 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× Sugahara, Keisuke× Suga, Tomo× Ootsuka, Yoshimi× Shiomi, Naoko× Iwakawa, Mayumi× Imai, Takashi× 道川 祐市× 菅原 圭亮× 菅 智× 荘司 好美× 塩見 尚子× 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 | (1) Background: Despite recent innovations in high throughput shotgun sequencing technologies, complex rearrangements in addition to the original dizygotic existence of homologous chromosomes complicate cancer genome sequencing. Thus there is an urgent need for establishing a convenient method to manipulate long stretches of individual chromosomes for analyzing sequence information. (2) Materials and Methods: A novel methodology has been developed to amplify single chromosomes for genotyping. A key feature of this methodology is a solid-phase multiple displacement amplification, that is an enzymatic reaction of Phi29 DNA polymerase, within a solidified agarose gel. It consists of the following seven steps. (I) Lysis of a limited number of cultured cells within a heated agarose gel solution to release chromosome molecules. (II) Careful aliquoting of small volumes of gel solutions containing limited number of chromosome molecules. (III) Solidification of the gel on ice. (IV) Solid-phase multiple displacement amplification of the gel-immobilized individual chromosome molecules. (V) Recovery of the amplification products by heating. (VI) Screening of target chromosomes by real-time QPCR. (VII) Multi-loci SNP typing using newly developed on-plastic chip allele-specific primer extension method (Michikawa et al., Anal. Sci., 2006, 22, 1537-1545). (3) Results: Utilization of agarose gel as a reaction matrix enabled reliable amplification-ready limited dilution of DNA to the level that homologous chromosomes rarely co-localize. Aggregation of chromosomes during the dilution step was dramatically reduced by incubating the gel solution at alkaline pH and at high temperature. Separation of homologous chromosomes by this method provided reliable determination of multi-loci genotypes on each homologous chromosome. Using this methodology, we have successfully determined haplotype of multiple SNPs in human ATM region that span 200 kilobase pairs. (4) Conclusions: The methodology developed in this study is effective for genotyping individual homologous chromosomes. Since amplified materials are easily recovered in a solution as PCR-ready form, this methodology is not restricted for genotyping. Further applications, such as chromosome-wide sequencing, are considerable. |
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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 | The 14th European Cancer Conference, ESTRO26 meeting, European Society for therapeutic radiology and oncology | |||||
| 発表年月日 | ||||||
| 日付 | 2007-09-27 | |||||
| 日付タイプ | Issued | |||||
