小基因
外显子捕获
外显子跳跃
外显子
剪接位点突变
外显子洗牌
RNA剪接
生物
剪接
分子生物学
遗传学
基因
选择性拼接
核糖核酸
作者
Lourdes R. Desviat,Belén Pérez,Magdalena Ugarte
出处
期刊:Methods in molecular biology
日期:2012-01-01
卷期号:: 37-47
被引量:31
标识
DOI:10.1007/978-1-61779-767-5_3
摘要
Although several bioinformatic tools exist to predict the effect on splicing of a nucleotide change, experimental verification with minigenes is essential for diagnostic purposes, as well as for revealing disease mechanisms and monitoring therapeutic interventions. Minigenes are splice reporter vectors (also known as exon-trapping vectors) that allow confirmation of the effect of mutations on the splicing process, indicated when patients' samples for RNA studies are not available. The minigene vector codes for exonic portions of a gene defined by functional 5′ splice donor and 3′ splice acceptor sites separated by intronic sequences where a polylinker is located. Here, the exon carrying the mutation under study is cloned along with its flanking intronic sequence. The resulting construct, in its wild-type and mutant sequence version, is transfected in established cell lines and the vector splicing pattern is analyzed. Ideally, the wild-type minigene results in correct exon inclusion, while the mutant construct results in exon skipping or other aberrant transcripts.
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