非同位素逆相点杂交方法检测苯丙酮尿症突变基因  

SCREENING MUTATIONS IN PHENYLKETONURIA BY MEANS OF NONRADIOACTIVE REVERSE DOT BLOT HYBRIDIZATION

作　　者：杨涛[1,2] 袁丽芳[1,2] 黄尚志[1,2] 赵时敏[1,2] 

机构地区：[1]中国医学科学院基础医学研究所中国协和医科大学基础医学院医学遗传室 [2]北京协和医院

出　　处：《中华医学遗传学杂志》1998年第5期307-309,共3页Chinese Journal of Medical Genetics

基　　金：中国医学科学院青年基金

摘　　要：目的建立一种简便、准确和快速的筛查苯丙酮尿症（ＰＫＵ）突变基因的方法。方法应用生物素渗入的聚合酶链反应扩增中国人ＰＫＵ患者常见突变位点：Ｙ２０４Ｃ（ｅｘｏｎ６，Ｅ６）、Ｒ２４３Ｑ（Ｅ７）、Ｙ３５６Ｘ（Ｅ１１）和Ｒ４１３Ｐ（Ｅ１２）所在的４个外显子区域，扩增标记产物再与固定于同一张膜上的等位基因特异性寡核苷酸探针进行逆相点杂交（ＲＤＢ），用碱性磷酸酶显色法检测杂交信号以确定突变类型。结果建立了检测以上突变位点的非同位素逆相点杂交方法，并对５例就诊的ＰＫＵ患者进行了ＲＤＢ检测，查明３例携带Ｒ２４３Ｑ突变，其中１例还带有Ｙ３５６Ｘ突变，用单链构象多态性方法验证了上述结果。Objective To establish a simple, accurate and rapid method for screening of the mutant genes in phenylketonuria (PKU).Methods Four exons harboring the mutations, Y204C(exon6, E6), R243Q(E7), Y356X(E11) and R413P(E12), were amplified by polymerase chain reaction (PCR) with incorporation of biotinylated deoxynucleotide(biotinylated 11 dUTP or biotinylated 14 dCTP). Hybridization between immobilized allele specific oligonucleotied probes and biotin labelled amplified DNA was performed and nonradioactively detected by a colorimetric reaction using streptavidin alkaline phosphatase.Results The methods of non radioactive reverse dot blot hybridization were established to screen the mutations. We detected the genotypes of five PKU patients and found that three of them carried R243Q mutation and one of the three also carried Y356X mutation. These results were confirmed by PCR single strand conformation polymorphism.Conclusions This method is suitable for rapid screening for common mutations in Chinese PKU patients.

关 键 词：苯丙酮尿症 苯丙氨酸羟化酶 逆相斑点杂交 PCR 

分 类 号：R589.304[医药卫生—内分泌]