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The development of DNA diagnosis by PCR and Capillary electrophoresis

PCR和毛细管电泳DNA诊断的进展

基本信息

批准号：
07672326
负责人：
ARAKAWA Hidetoshi
金额：
$ 1.22万
依托单位：
Showa University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07672326/
关键词：
Capillary electrophoresis DNA diagnosis PCR Double strand DNA SSCP 日本鎖DNA 2本鎖DNA

项目摘要

Capillary electrophoresis (CE) was studied for the analysis of PCR amplified sample. Alowcross linked polyacrylamide gel and entangled polymer solution (polymer solution) were used for CE.Laser induced fluorescence (LIF) detection was performed using Thiazole orange as the fluorescent intercalating dye. The highly sensitive LIF-CE was applied to detection of allele specific PCR for medium-chain acyl-CoA dehydrogenase deficiency and phenylketonuria mutation, and of PCR-restriction fragment length polymorphism for mutant dive E gene. Analysis of single strand conformation polymorphism (SSCP) by CE was also developed. The conformational change of the single strand DNA is caused by a mutation in DNA fragment. The change is detected as mobility shift on CE.The effects of acrylamide gel concentration, running-temperature and fragment size amplified by PCR were studied to develop the separation of SSCP.The results obtained in this report showed that CE is well suited for clinical DNA analysis using PCR.Analysis of SSCP by laserinduced fluorescence capillary electrophoresis in entangled polymer solution (CE-LIF) has benn developed also in this study. K-ras genes including seven mutations were amplified with primer labeled with Texas Red at its 5' end. The labeled PCR products were separated with capillary gel electrophoresis and He-Ne laser-excited fluorescence detection. All fragments having normal (Gly) and mutated (Ala, Arg, Cys, Ser, Val, Asp) sequences at codon 12 can be distinguished by this method. Further more, SSCP analysis was carried out with multiple sheath-flow gel capillary-array electrophoresis to analyze many samples simultaneously. CE-LIF is well suited for clinical analysis of SSCPs because of its high sensitivity, resolution, reproducibility and speed.

采用毛细管电泳（CE）技术对扩增后的样品进行分析。使用允许交联聚丙烯酰胺凝胶和纠缠聚合物溶液（聚合物溶液）进行CE。以噻唑橙为荧光插层染料进行激光诱导荧光检测。应用高灵敏度的liff - ce检测中链酰基辅酶a脱氢酶缺乏症和苯丙酮尿突变的等位基因特异性PCR，以及突变体dive E基因的PCR限制性片段长度多态性。应用CE对单链构象多态性（SSCP）进行了分析。单链DNA的构象变化是由DNA片段的突变引起的。这种变化是通过CE的迁移率变化来检测的。研究了丙烯酰胺凝胶浓度、运行温度和PCR扩增片段大小对SSCP分离的影响。本报告的结果表明，CE非常适合用于PCR的临床DNA分析。本研究还开发了纠缠聚合物溶液中激光诱导荧光毛细管电泳（CE-LIF）分析SSCP的方法。K-ras基因包括7个突变，引物在其5'端标记为Texas Red。标记的PCR产物用毛细管凝胶电泳和He-Ne激光激发荧光检测分离。所有在密码子12处具有正常（Gly）和突变（Ala, Arg, Cys, Ser, Val, Asp）序列的片段都可以通过该方法进行区分。采用多套鞘流凝胶毛细管阵列电泳进行SSCP分析，可同时分析多个样品。CE-LIF具有高灵敏度、高分辨率、高重现性和高速度等优点，非常适合sscp的临床分析。