优化PCR体系结合毛细管电泳技术检测FMR1基因前突变与全突变

目的 通过优化PCR并结合毛细管电泳,建立高扩增效率、高分辨率的FMR1基因CGG重复序列异常扩增检测体系.方法 选择标准样本和经Southern印迹技术确定(CGG)n的正常、前突变、全突变男性和女性样本15例,进行PCR检测体系的优化.优化的PCR扩增产物经琼脂糖凝胶电泳、聚丙烯酰胺凝胶电泳和毛细管电泳等多种方法进行结果比较.结果 经优化的PCR体系可以检测出(CGG)n大于260个拷贝的全突变男性和(CGG)n达到183个拷贝的前突变女性.毛细管电泳能够清晰分辨出相差1个CGG的两个等位基因,结果具有良好的可重复性.结论 该PCR检测体系大幅度提高了普通PCR方法的扩增效率和分辨率,明显降低了对于Southern印迹技术的依赖,可以作为临床筛查FMR1基因突变的首选方法.

Optimization of polymerase chain reaction assay combined with capillary electrophoresis to detect the pre- and full mutation of FMR1 gene

Objective To develop an efficient, high resolution PCR assay suitable for detection of the (CGG)n repeats of the fragile X mental retardation 1 (FMR1)gene by optimizing the PCR system in combination with capillary electrophoresis. Methods Three standard samples and twelve samples that were verified by Southern blot analysis including both male and female in the normal, pre- and full mutation range were used in this study to evaluate the enhanced PCR system. All amplicons were electrophoresed by agarose, polyacrylamide and capillary electrophoresis to compare the results. Results The enhanced PCR assay developed in this study was able to detect a full mutation with (CGG)n being larger than 260 repeats in a male. An expanded premutation allele with (CGG)n as large as 183 repeats in a female was also amplified.The capillary electrophoresis method used in this study was able to distinguish two alleles with 1 CGG repeat difference and the results were reproducible. Conclusion A high resolution PCR assay is developed, which is much more efficient than the general PCR systems. It is suitable for the clinical screening of FMR1 gene and will greatly reduce the number of Southern blot analysis needed in clinical application.