Use of splicing reporter minigene assay to evaluate the effect on splicing of unclassified genetic variants

Methods Mol Biol. 2010;653:249-57. doi: 10.1007/978-1-60761-759-4_15.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Review

MeSH terms

  • Alternative Splicing / genetics
  • Alternative Splicing / physiology*
  • Cells, Cultured
  • Genes, Reporter*
  • Genetic Diseases, Inborn / diagnosis*
  • Genetic Diseases, Inborn / genetics
  • Genetic Diseases, Inborn / metabolism
  • Genetic Techniques
  • Genetic Variation / physiology*
  • Humans
  • Neoplasms / diagnosis
  • Neoplasms / genetics
  • Neoplasms / metabolism
  • RNA, Messenger / analysis
  • RNA, Messenger / genetics*
  • RNA, Messenger / metabolism*
  • Transfection / methods


  • RNA, Messenger