应用双脱氧指纹法检测苯丙氨酸羟化酶基因突变

DETECTION OF THE MUTANT PHENYLALANINE HYDROXLASE GENE BY DIDEOXY FINGERPRINTING

目的提高苯丙氨酸羟化酶基因突变的检出效率，快速对苯丙酮尿症患儿进行产前诊断。方法采用双脱氧指纹（ｄｉｄｅｏｘｙｆｉｎｇｅｒｐｒｉｎｔｉｎｇ，ｄｄＦ）检测技术，ｄｄＦ法是将单链构象多态（ｓｉｎｇｌｅｓｔｒａｎｄｃｏｎ－ｆｏｒｍａｔｉｏｎｐｏｌｙｍｏｒｐｈｉｓｍ，ＳＳＣＰ）分析技术和双脱氧测序技术结合为一体的检测基因突变的方法，它能有效地检测基因突变而不受扩增片段长度的限制。用ｄｄＦ法鉴定了苯丙氨酸羟化酶基因上的突变Ｙ１６５Ｘ，Ｙ２０４Ｃ和Ｑ３５５Ｈ。结果有多条泳带不同于正常对照泳带。采用ＳＳＣＰ技术，也检测出突变Ｙ１６５Ｘ和Ｑ３５５Ｈ显示了不同于正常的泳带位移，而扩增片段较长的含突变Ｙ２０４Ｃ的样本却未显示异常泳带。结论ｄｄＦ检测技术对大片段基因的突变检测比ＳＳＣＰ法更为敏感。

Objective To establish a method with high efficiency in detecting phenylalanine hydroxylase(PAH) gene mutations and hence to rapidly diagnose prenatal fetals with phenylketonuria. Methods Dideoxy fingerprinting (ddF) was used.It is a hybrid method of dideoxy sequencing and single strand conformation polymorphism(SSCP);it can effectively detect the presence of mutant genes and would not be limited by the length of the amplified products,by ddF,the mutant genes Y165X,Y204C and Q355H of PAH genes were detected. Results Many bands showed altered migration,compared with the controls.Abnormal band migration in Y165X and Q355H was also detected by SSCP,but it was not detected in Y204C which had large amplified products. Conclusion Compared with SSCP, ddF is a more sensitive method detecting gene mutations in large fragments,ddF is a practicable method for detecting gene mutations.