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目的:选取本实验室筛查SCN1A突变的Dravet综合征患者,通过生物信息学方法分析其对剪接的影响,构建迷你基因进行体外剪接分析。方法:使用Human Splicing Finder数据库预测c.1738C>T突变对剪接的潜在影响,通过PCR方法扩增SCN1A第10、11、12、13号外显子及两端部分内含子,克隆到pTARGET真核表达载体,构建pTARGET-EXON-10-11-12-13迷你基因,以野生型pTARGET-EXON-10-11-12-13为模板构建c.1738C>T突变体。将野生型和c.1738C>T突变体分别转染HEK293细胞,提取总RNA进行RT-PCR。结果:野生型与c.1738C>T突变体RT-PCR产物大小都为591bp。结论:c.1738C>T突变不是通过对剪接的影响导致疾病发生。
Objective: To select the patients with Dravet syndrome who screened for SCN1A mutation in our laboratory and analyze their effects on splicing by bioinformatics methods, and construct minigene for in vitro splicing analysis. Methods: Human Splicing Finder database was used to predict the potential impact of c.1738C> T mutation on splicing. The exon 10, 11, 12 and 13 exons of SCN1A and some intron were amplified by PCR and cloned into pTARGET eukaryotic Expression vector. The pTARGET-EXON-10-11-12-13 minigene was constructed and the c.1738C> T mutant was constructed by using the wild-type pTARGET-EXON-10-11-12-13 as a template. The wild-type and c.1738C> T mutants were transfected into HEK293 cells, and the total RNA was extracted for RT-PCR. Results: RT-PCR products of both wild type and c.1738C> T mutant were 591bp in size. Conclusions: The c.1738C> T mutation does not result in the onset of disease by its effect on splicing.