多重PCR-SSCP检测人瘢痕疙瘩Fas基因突变

目的　建立多重PCR -SSCP反应体系 ,以筛选瘢痕疙瘩Fas基因突变。方法　按照多重PCR引物设计原则设计相应引物 ,经分析、实验得到理想的多重PCR扩增条件 ,以此条件扩增目的基因 ;将所得PCR反应产物在单基因对照下进行银染多重SSCP分析 ,进而筛选基因突变。结果　外显子 6 ,8,9的单一PCR反应产物及多重PCR反应产物经琼脂糖凝胶电泳 ,各条带清晰 ,无非特异性扩增 ,且产量较高 ,产物的长度与理论值相符 ;经银染多重SSCP分析 ,正常皮肤组织和增生性瘢痕中均未检出突变 ,在 2 3例瘢痕疙瘩标本中 ,18例出现异常电泳带。结论　本实验建立了用以分析瘢痕疙瘩Fas基因突变的多重PCR -SSCP方法 ,分析结果提示 :瘢痕疙瘩组织中Fas基因的突变可能与该病的发生有密切关系 ;多重PCR -SSCP是检测基因点突变的一种快速、敏感、有效的方法

Detection of Fas gene mutations in human keloids by mPCR-SSCP analysis

Objective To establish a mPCR-SSCP technique to detect the mutant exons of Fas gene in keloids. Methods Three pairs of primers designed according to the principle of mPCR. After every fragment was amplified successfully, the appropriate condition of multiplex PCR was determined. The mPCR products were analyzed by mSSCP with silver stain with the control of single exon SSCP to detect gene mutations. Results The bands of three products were seen in agarose gel electrophoresis clearly and unique without nonspecific amplified fragment, the DNA sequences of these three bands were in coincidence with lengthes and sequences which had been predicted. By mPCR-SSCP analysis shifts in electrophoretic mobility of the Fas gene were detected in 18 of 23 keloids,but none in all hypertrophic scars and normal skin tissue. Conclusion An economical and fast mPCR-SSCP method for detection of Fas gene mutations in keloids were established; the results indicated the mutations of Fas gene could play an important role in pathogenesis of keloids.