The development of multiple SNPs typing system by flow-arry

Flow-arry多SNP分型系统的开发

• 批准号: 14370071
• 负责人: SASAKI Kohsuke
• 金额: $ 8.45万
• 依托单位: Yamaguchi University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14370071/
• 关键词: DNA; SNP; Flow cytometry (FCM); Hybridization; fluorescence; high-throughput analysis; drug response; genotype; フローサイトメトリー; ゲノム; フローアレイ; 蛍光; SNPs

DNA sequence variation is associated with a certain disease and differential drug response. Single nucleotide polymorphisms (SNPs) are common type of variant. In this project, we have developed a new method for high-throughput genotyping of SNPs with microspheres of various sizes and a flow cytometer equipped with only a single laserThis system uses microspheres of various sizes with oligonucleotide probes attached by covalent bonds. Polymerase chain reaction (PCR) products labeled with Alexa 488 are hybridized to the probes and then hybridization signals are analyzed by flow cytometry. In order to type a specific SNP, two kinds of beads are bound to paired probes ; one complementary to the wild-type sequence and the other to the variant sequence. The paired beads are placed in the same tube and the number of normal beads and variant beads ratio are three to one. When the beads with higher fluorescence intensity are about three times as many as lower ones, this sample is determined wild-type genotype. When multiplex analysis is carried out, the beads for each SNP site is identified by their diameters. In this project, we have developed a novel method in which SNP genotyping with microspheres of various sizes and flowcytometer equipped with single laser. The method increases parameters in beads analysis

DNA序列变异与某种疾病和不同的药物反应有关。单核苷酸多态(SNPs)是常见的变异类型。在这个项目中，我们开发了一种新的高通量SNPs基因分型方法，使用不同大小的微球和只配备单个激光的流式细胞仪。该系统使用不同大小的微球和以共价键连接的寡核苷酸探针。用Alexa 488标记的聚合酶链式反应(PCR)产物与探针杂交，然后用流式细胞仪分析杂交信号。为了对特定的SNP进行分型，两种珠子与成对的探针结合；一种与野生型序列互补，另一种与变异序列互补。将成对的珠子放置在同一管中，法珠数和变珠率为3：1。当荧光强度较高的珠子大约是荧光强度较低的珠子的三倍时，该样本被确定为野生型基因。当进行多重分析时，每个SNP位点的珠子通过它们的直径来识别。在这个项目中，我们开发了一种新的方法，用不同尺寸的微球和配备了单激光的流式细胞仪进行SNP基因分型。该方法增加了珠子分析中的参数

项目成果

Harada T, Okita K, Shiraishi K, Kusano N, Furuya T, Oga A, Kawauchi S, Kondoh S, Sasaki K.: "Detection of genetic alterations in pancreatic cancers by comparative genomic hybridization coupled with tissue microdissection and degenerate oligonucleotide pri

Harada T、Okita K、Shiraishi K、Kusano N、Furuya T、Oga A、Kawauchi S、Kondoh S、Sasaki K.：“通过比较基因组杂交结合组织显微切割和简并寡核苷酸检测胰腺癌的遗传改变

Furuya T.: "A novel technology allowing immunohistochemical staining of a tissue section with 50 different antibodies in a single experiment"J Histochem Cytochem. 52・2. 205-210 (2004)

Furuya T.：“一种允许在一次实验中使用 50 种不同抗体对组织切片进行免疫组织化学染色的新技术”J Histochem Cytochem 52・2 (2004)。

Takita M.: "An analysis of changes in the expression of cyclins A and B1 by the cell array system during the cell cycle : comparison between cell synchronization methods"Cytometory. 55A・1. 24-29 (2003)

Takita M.：“细胞周期期间细胞阵列系统对细胞周期蛋白A和B1的表达变化的分析：细胞同步方法之间的比较”55A·1（2003）。

Kawasaki K.: "11q23-24 loss is associated with chromosomal instability in endometrial cancer"Int J Mol Med.. 12・5. 727-731 (2003)

川崎 K.：“11q23-24 缺失与子宫内膜癌的染色体不稳定有关”Int J Mol Med.. 12・5 (2003)。

Harada T.: "Interglandular cytogenetic heterogeneity detected by comparative genomic hybridization in pancreatic cancer"Cancer Research. 62(3). 835-839 (2002)

Harada T.：“通过比较基因组杂交检测胰腺癌的腺间细胞遗传学异质性”癌症研究。