CAJ | 학술논문

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.
목적 통과우화PCR병결합모세관전영,건립고확증효솔、고분변솔적FMR1기인CGG중복서렬이상확증검측체계.방법 선택표준양본화경Southern인적기술학정(CGG)n적정상、전돌변、전돌변남성화녀성양본15례,진행PCR검측체계적우화.우화적PCR확증산물경경지당응효전영、취병희선알응효전영화모세관전영등다충방법진행결과비교.결과 경우화적PCR체계가이검측출(CGG)n대우260개고패적전돌변남성화(CGG)n체도183개고패적전돌변녀성.모세관전영능구청석분변출상차1개CGG적량개등위기인,결과구유량호적가중복성.결론 해PCR검측체계대폭도제고료보통PCR방법적확증효솔화분변솔,명현강저료대우Southern인적기술적의뢰,가이작위림상사사FMR1기인돌변적수선방법.
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.