LDL受体基因内含子15结构分析及其在家族性高胆固醇血症基因诊断中的应用  

The Sequence Analysis of the 3' End of Intron 15 from Human LDL Receptor Gene and Its Application in the Study of Gene Diagnosis for Familial Hypercholesterolaemia

作　　者：周天鸿[1] 李月琴[1] 刘飞鹏[1] 海戎[1] 朱嘉明[1] 孙宏[2] 

机构地区：[1]暨南大学生物工程系,广州510632 [2]暨南大学医学院附属医院,广州510630

出　　处：《Acta Genetica Sinica》1999年第1期1-7,共7页

基　　金：国务院侨办重点学科科研基金;广东省自然科学基金!950644

摘　　要：为了解人类 ＬＤＬ受体基因内含子 １５的遗传背景，利用长链 ＰＣＲ和锚定 ＰＣＲ分离了 ＬＤＬ受体基因外显子１５－内含子１５－外显子１６和内含于１５的３’末端片段。利用Ｄｙｎａｌｂｅａｄｓ 固相单链分离ＰＣＲ产物直接测序法测定了内含子１５ ３’末端１２２２个碱基序列。序列显示：３’ 末端含有由１６个碱基组成的典型３’末端剪接位点；３’端上游第３１个碱基处含有经典分支位 点。除了经典分支位点外，在３’末端上游第２０个碱基处，还含有一个可能的隐蔽分支位点。 序列还显示了被认为与血清胆固醇水平相关的ＰｖｕⅡ酶切多态性的碱基变异性质，即碱基由 Ｃ－Ｔ的改变同时利用所测的序列，设计了相应的引物，建立了ＰＣＲ检测ＬＤＬ受体基因内含 子 １５ ＰｖｕⅡ多态性技术，并证实此技术可以快速、简便地应用到家族性高胆固醇血症的基因 诊断上。To understand intron 15 of human LDL receptor gene, the DNA fragments from exon 15 to exon 16 and the 3' end of intron 15 were amplified with long chain PCR and anchored PCR. The 3' end of intron 15 was sequenced with Dynalbeads Streptavidin Solid Phase technique. The sequence analysis showed that the 3' end of intron 15 contained the 3' splicing site and the branch site at 31 nucleotides upstream of the 3' end. Besides the authentic branch site, it is possible that the 3' end of intron 15 contained a cryptic site (GCCTCAC) at 20 nucleotides upstream of the 3' end. The sequences suggest that the PvuII polymorphism at intron 15 is caused by the T-C substitution. According to the sequences of the 3' end, the new PCR-RFLP protocol for detection of PvuII polymorphism at intron 15 was developed. Using this protocol a representative familial hypercholesterolaemia family was identified with linkage analysis of PvuII polymorphism at intron 15.

关 键 词：LDL受体基因 序列分析 高胆固醇血症 RFLP PCR 

分 类 号：R589.2[医药卫生—内分泌]