Development of automated gene diagnoses using capillary electrophoresis

使用毛细管电泳进行自动化基因诊断的开发

• 批准号: 07557017
• 负责人: YUASA Yasuhito
• 金额: $ 9.98万
• 依托单位: Tokyo Medical and Dental University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07557017/
• 关键词: capillary electrophoresis; gene diagnosis; PCR; mutation; microsatellite; colorectal cancer; SSCP

We developed a method to analyze a polyadenine tract, the (A)_<10> repeat, within the cysteine-rich domain of the transforming growth factor-beta (TGF-beta) type II receptor gene using a non-gel-sieving capillary electrophoresis technique and applied it to the DNA diagnosis of colorectal cancers. This method consists of single-strand DNA amplification of the (A)_<10> repeat by an asymmetric PCR technique and capillary electrophoresis. A higher concentration of dATP in the PCR reaction mixture led to more specific amplification of the (A)_<10> repeat. Under the optimal electrophoretic conditions, one nucleotide difference could be determined in 8 to 32 nucleotides. One or two base deletions of the (A)_<10> repeat in colorectal cancers could be detected under these conditions within 30 min, and the results coincided with those obtained on DNA sequencing analyzes. According to a sensitivity study, we could detect the deleted sequence if it was present in 12.5% or more of the wild-type allele. The reproducibility of this technique was satisfactory because the intraassay imprecision (CV) (n=10) was 1.4%. These results indicate that capillary electrophoretic analysis of small repeated sequences results in easier handling and more feasible automation, com-pared with conventional gel electrophoretic analysis.

我们开发了一种方法来分析的多聚腺嘌呤道，（A）_<10>重复序列，在富含半胱氨酸的结构域的转化生长因子-β（TGF-β）II型受体基因使用非凝胶筛分毛细管电泳技术，并将其应用于大肠癌的DNA诊断。该方法包括<10>通过不对称PCR技术和毛细管电泳对（A）_重复序列进行单链DNA扩增。PCR反应混合物中较高浓度的dATP导致（A）_重复序列的更特异性扩增<10>。在最佳电泳条件下，可在8至32个核苷酸中确定一个核苷酸差异。在<10>此条件下，30 min内即可检测到大肠癌中（A）_重复序列的1 ~ 2个碱基缺失，其结果与DNA测序结果一致。根据敏感性研究，如果缺失序列存在于12.5%或更多的野生型等位基因中，则我们可以检测到缺失序列。该技术的重现性令人满意，因为批内不精密度（CV）（n=10）为1.4%。这些结果表明，毛细管电泳分析小重复序列与常规凝胶电泳分析相比，操作简便，自动化程度高。

项目成果

湯浅 保仁: "無敵のバイオテクニカルシリーズ PCR実験ノート DNA多型,変異の解析" 谷口武利編, 12 (1997)

汤浅泰仁：《无敌生物技术系列PCR实验笔记DNA多态性和突变的分析》谷口武敏编辑，12（1997）

Lu,S.-L.,et al.: "Loss or somatic mutations of hMSH2 occur in hereditary nonpolyposis colorectal cancers with hMSH2 germline mutations." Jpn.J.Cancer Res.87. 279-287 (1996)

Lu,S.-L.,et al.：“hMSH2 丢失或体细胞突变发生在具有 hMSH2 种系突变的遗传性非息肉病性结直肠癌中。”

Arai T,Akiyama Y,Okabe S,Ando M,Endo M,Yuasa Y.: "Genomic structure of the human Smad3 gene and its infrequent alterations in colorectal cancers." Cancer Letters. 122. 157-163 (1998)

Arai T、Akiyama Y、Okabe S、Ando M、Endo M、Yuasa Y.：“人类 Smad3 基因的基因组结构及其在结直肠癌中的罕见改变。”

Arai,T.,et al.: "Genomic structure of the human Smad3 gene and its infrequent alterations in colorectal cancers." Cancer Letters. 122. 157-163 (1998)

Arai,T.,et al.：“人类 Smad3 基因的基因组结构及其在结直肠癌中的罕见改变。”

Oto M, er al.: "Analysis of polyadenine tract of the TGF-β type II receptor gene in colorectal cancers by non-gel sieving capillary electrophoresis" Clinical Chemistry. 43. 759-763 (1997)

Oto M 等人：“通过非凝胶筛分毛细管电泳分析结直肠癌中 TGF-β II 型受体基因的聚腺嘌呤束”，临床化学 43. 759-763 (1997)。