脆性X综合征致病基因FMR1一个新的外显子及其鉴定-临床检验诊断学专业论文.docxVIP

福建医科大学 福建医科大学 2008 级硕士研究生毕业论文 5 5 At the same time, we used hybrid minigene splicing assay to analyse the novel alternative exon at cellular level. We used PCR to amplify the minigene (intron 9 of FMR1 gene and its adjacent exon 9 and exon 10) from the DNA of a suspected FXS patient and a norml control, and the PCR products were inserted into the pcDNATM3.1 / Hygro (+) vector. 48 hours after transfection of HeLa cells, total RNA was extracted, followed by reverse transcription and semi-nested PCR. Sequencing of the inserted segment from the patient and the normal control showed that there were three base changes: 21452 C → T, 21513 C → T and 23784 T → C, comparied to reference sequence of FMR1 in GenBank. The first two base changes were polymorphic sites, where 23784 T → C might lead to an ISE based on bioinformatical analysis. The first two base changes were also present in the insert of control. Nevertheless, semi-nested PCR and the sequencing of its products showed that the novel exon in the processed transcripts of HeLa cells transfected by of recombinant vectors from both the patient and the normal control was detectable. The present study verified the presence of a novel criptic alternative exon in the intron 9of human FMR1 gene, and laid foundation to study the relationship of FMR1 and FXS pathogenesis. Keywords: FMR1; alternative splice; bioinformatics; exon; semi-nested PCR 福建医科大学 福建医科大学 2008 级硕士毕业论文 1 1 英文缩略词表 ABBREVIATIONS 缩写 英 文 全 称 中 文 全 称 FXS fragile X syndrome 脆性 X 综合征 FMR1 fragile X mental retardation 1 gene 脆性 X 智力低下 1 基因 FMRP fragile X mental retardation protein 脆性 X 智力低下蛋白 PCR polymerase chain reaction 聚合酶链反应 cDNA complementary deoxyribonucleic acid 互补 DNA RT reverse transcription 逆转录 ddH2O double distilled H2O 双蒸水 DNA deoxyribonucleic acid 脱氧核糖核酸 dNTP deoxyribonucleoside triphosphate 脱氧三磷酸核苷 bp base pair 碱基对 kD kilodalton 千道尔顿 LB Luria Bertani medium LB 培养基 DEPC diethyl pyrocarbonate 焦碳酸二乙酯 min minute 分钟 PBS phosphate-buffered saline 磷酸盐缓冲生理盐水