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Use of splicing reporter minigene assay to evaluate the effect on splicing of unclassified genetic variants.
- Source :
-
Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2010; Vol. 653, pp. 249-57. - Publication Year :
- 2010
-
Abstract
- 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 mRNA 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.
- Subjects :
- Alternative Splicing genetics
Cells, Cultured
Genetic Diseases, Inborn genetics
Genetic Diseases, Inborn metabolism
Genetic Techniques
Humans
Neoplasms diagnosis
Neoplasms genetics
Neoplasms metabolism
RNA, Messenger analysis
Transfection methods
Alternative Splicing physiology
Genes, Reporter
Genetic Diseases, Inborn diagnosis
Genetic Variation physiology
RNA, Messenger genetics
RNA, Messenger metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1940-6029
- Volume :
- 653
- Database :
- MEDLINE
- Journal :
- Methods in molecular biology (Clifton, N.J.)
- Publication Type :
- Academic Journal
- Accession number :
- 20721748
- Full Text :
- https://doi.org/10.1007/978-1-60761-759-4_15