Takada-Matsuzaki Y, Mukaida M
Department of Forensic Medicine, National Defense Medical College, Tkorozawa, Japan.
Nihon Hoigaku Zasshi. 1999 Nov;53(3):330-7.
Constant Denaturing Gel Electrophoresis (CDGE) was used as an analytical method of the genetic markers. Even a single base change in a fragment amplified by PCR was detected exactly by CDGE. The computational simulation of CDGE gave the calculation whether a single base change in a fragment amplified by PCR could be detected by CDGE or not. In this report, genotyping of three blood groups, MN, Duffy and Kidd, and Gc system is described. The regions reflecting allelic differences of each system were amplified from genomic DNAs. The concentration of denaturants (urea and formamide) in CDGE gels was decided with the computational simulation as follows: 15% for MN, 27% for Duffy, 24% for Kidd, 30% for Gc. CDGE was run in TBE buffer at 60 degrees C, 100 V constant voltage. PCR amplified fragments with 1-3 base changes were separated clearly in each gel. By staining the gels with ethidium bromide, the genotype of each system was determined. The genotyping of system by CDGE can avoid mistakes in the conventional method, which requires complicated and multiple troublesome operations. Analysis of PCR amplified fragments by CDGE will make a beneficial contribution to medico-legal practice.
恒定变性凝胶电泳（CDGE）被用作遗传标记的分析方法。通过CDGE能够准确检测出经聚合酶链式反应（PCR）扩增片段中哪怕仅一个碱基的变化。CDGE的计算模拟可得出经PCR扩增的片段中单个碱基变化能否被CDGE检测到的计算结果。在本报告中，描述了MN、达菲和基德三种血型以及Gc系统的基因分型。从基因组DNA中扩增出反映每个系统等位基因差异的区域。CDGE凝胶中变性剂（尿素和甲酰胺）的浓度通过计算模拟确定如下：MN为15%，达菲为27%，基德为24%，Gc为30%。CDGE在TBE缓冲液中于60摄氏度、100V恒定电压下运行。在每种凝胶中，具有1至3个碱基变化的PCR扩增片段都能被清晰分离。通过用溴化乙锭对凝胶进行染色，确定每个系统的基因型。通过CDGE对系统进行基因分型可避免传统方法中需要复杂且多次繁琐操作而产生的错误。利用CDGE对PCR扩增片段进行分析将对法医实践做出有益贡献。
