Use of Splicing Reporter Minigene Assay to Evaluate the Effect on Splicing of Unclassified Genetic Variants
Use of Splicing Reporter Minigene Assay to Evaluate the Effect on Splicing of Unclassified Genetic Variants
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DOI：
10.1007/978-1-60761-759-4_15
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发表时间：
2010-01-01
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影响因子：
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通讯作者：
Tosi, Mario
中科院分区：
文献类型：
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作者：
Gaildrat, Pascaline;Killian, Audrey;Tosi, Mario
对基因筛查中发现的众多具有未知生物学和临床意义的序列变异（UV 表示“未分类变异”）的解释是遗传疾病（包括癌症易感性）分子诊断中的重大挑战。一小部分 UV 可能是有害的，因为它们会影响 mRNA 剪接。在这里，我们描述了一种基于小基因构建体的功能剪接测定，该测定评估序列变异对剪接的影响。从患者基因组 DNA 中 PCR 扩增包含感兴趣的变异序列以及侧翼内含子序列的基因组片段，并将其克隆到小基因载体中。瞬时转染培养细胞后，通过逆转录 PCR 分析和测序比较野生型和变异构建体产生的转录物的剪接模式。该方法代表了对患者 RNA 进行逆转录 PCR 分析的补充方法，用于鉴定致病性剪接突变。
The interpretation of the numerous sequence variants of unknown biological and clinical significance (UV for "unclassified variant") found in genetic screenings represents a major challenge in the molecular diagnosis of genetic disease, including cancer susceptibility. A fraction of UVs may be deleterious because they affect m RNA splicing. Here, we describe a functional splicing assay based on a minigene construct that assesses the impact of sequence variants on splicing. A genomic segment encompassing the variant sequence of interest along with flanking intronic sequences is PCR-amplified from patient genomic DNA and is cloned into a minigene vector. After transient transfection into cultured cells, the splicing patterns of the transcripts generated from the wild-type and from the variant constructs are compared by reverse transcription-PCR analysis and sequencing. This method represents a complementary approach to reverse transcription-PCR analyses of patient RNA, for the identification of pathogenic splicing mutations.