The development of DNA diagnosis by PCR and Capillary electrophoresis

采用毛细管电泳法对扩增后的样品进行分析。采用低交联聚丙烯酰胺凝胶和缠绕聚合物溶液(聚合物溶液)进行毛细管电泳法，以噻唑橙为荧光插层染料进行激光诱导荧光检测。应用高灵敏的LIF-CE检测中链酰辅酶A脱氢酶缺乏症和苯丙酮尿症的等位基因特异性聚合酶链式反应，以及突变的DIVE基因的聚合酶链式反应-限制性片段长度多态性。建立了单链构象多态(SSCP)的CE分析方法。单链DNA的构象变化是由DNA片段的突变引起的。研究了丙烯酰胺凝胶浓度、运行温度和聚合酶链式反应扩增片段大小对凝胶电泳法分离SSCP的影响。结果表明，毛细管电泳法非常适用于临床DNA分析。用德克萨斯红标记的5‘端引物扩增包括7个突变的K-ras基因。用毛细管凝胶电泳法和He-Ne激光激发荧光检测法分离标记的聚合酶链式反应产物。所有密码子12处具有正常(Gly)和突变(Ala、Arg、Cys、Ser、Val、Asp)序列的片段都可以用这种方法区分。采用多鞘流凝胶毛细管阵列电泳法进行SSCP分析，可同时分析多个样品。CE-LIF具有灵敏度高、分辨率高、重复性好、快速等优点，非常适合临床应用。

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.