The development of DNA diagnosis by PCR and Capillary electrophoresis

PCR和毛细管电泳DNA诊断的进展

• 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; Capillary electrophoresis; DNA diagnosis; PCR; Double strand DNA; SSCP; 日本鎖DNA; 2本鎖DNA

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.

研究了毛细管电泳（CE）以分析PCR扩增样品。使用甲基丙烯酰胺凝胶和纠缠聚合物溶液（聚合物溶液）进行CE。激光器诱导的荧光（LIF）检测使用噻唑橙作为荧光插入染料。高度敏感的LIF-CE用于检测等位基因特异性PCR中的中链酰基-COA脱氢酶缺乏症和苯基酮尿突变，以及突变潜水E基因的PCR限制片段长度多态性。还开发了CE分析单链构象多态性（SSCP）。单链DNA的构象变化是由DNA片段突变引起的。 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这项研究。 K-RAS基因在内的K-RAS基因在其5'末端用德克萨斯红色标记的底漆进行扩增。用毛细管凝胶电泳和HE-NE激光激发荧光检测将标记的PCR产物分离。所有具有正常（Gly）和突变（Ala，arg，cys，ser，val，asp）序列的片段可以通过这种方法区分。此外，使用多个护鞘凝胶毛细管阵列电泳进行SSCP分析，以同时分析许多样品。 CE-LIF非常适合SSCP的临床分析，因为其敏感性高，解决性，可重复性和速度。