PCR－SSCP联合序列分析法在RB1基因突变检测中的应用

DETECTION OF MUTATIONS IN THE BR1 GENE WITH THE METHOD OF PCR-SSCP COMBINED SEQUENCING

用聚合酶链反应－单链构象多态（ＰＣＲ－ＳＳＣＰ）联合序列分析，对视网膜母细胞瘤肿瘤组织ＲＢ１基因存在状态进行检测。对ＲＢ１基因全部２７个外显子分别进行ＰＣＲ扩增后，选用适当的限制性核酸内切酶将扩增产物切割成２００ｂｐ左右的片段，加热至双链解离后，６％聚丙烯酰胺凝胶电泳。电泳后对有异常电泳迁移率条带所属标本的同一外显子行ＰＣＲ扩增、序列分析，进一步证实突变的位置和类型。本研究发现包括点突变、缺失、插入等ＲＢｌ基因改变，显示Ｐａｉｔ－ＳＳＣＰ联合序列分析在已知序列基因突变检测中的敏感性和可靠性。

owell JK2 With the method of PCR-SSCP combined sequening, we analysed the mutations in the mutations in RB1 geneof 10 retinoblastoma samples. All 27 exons of RB1 gene have been examined, The PCR preducts weredigested with some restriction endonuclease to the size of abotut 200 bp and then denatured into singlestrands Loading the subgle strands of DNA into the 6% polyacrilamide gel, we followed the technology named SSCP to find the abnormal bands in the SSCP gel.After that,the samples foundwith the abnormal bands in the SSCP gel were amplified again and sequenced to confirm the types andlocations of the mutations u1 the RB1 gene, We have fonnd sonie kinds of mutations in the RB1gene.