A high proportion of pathogenic variants in disease-responsible genes alter pre-mRNA processing or splicing ( Sanz et al 2010). More than 50% of DNA variants of susceptibility genes are of unknown clinical significance, which represent a serious problem for the genetic counselling of such diseases. 

Functional analysis of DNA variants from disease-responsible genes can be carried out by direct patient RNA RT-PCR (usually lymphocyte RNA) or functional assay by hybrid minigenes. 

Patient RNA is not always available and often difficult to obtain so splicing reporter minigenes are useful alternative tools to study ex vivo the impact of a variant on splicing. 

The Splicing and Genetic susceptibility group of the IBGM (CSIC, PI Eladio A. Velasco) has developed a new splicing reporter plasmid (pSAD) to carry out splicing functional assays by hybrid minigenes, which helps to identify variants with impact on splicing. 

The protocol for a splicing functional analysis of a particular DNA variant of a disease-responsible gene is as simple as follows:

1. Cloning of exon(s) of interest in pSAD and flanking introns. It is recommended to keep the genomic context with 5’ and 3’ exons. 

2. Generation of the DNA variant by site directed-mutagenesis.

3. Transfection of the wild type and mutant minigenes into eukaryotic cells.

4. RNA Isolation and RT-PCR with specific primers of the constitutive exons (V1 and V2) that do not amplify RNA transcribed by the host cell.

5. Electrophoresis and sequencing of RNA isoforms: characterization of anomalous events (e.g. exon skipping, use of alternative splice sites, intron retention, etc).

The full protocol (cloning and functional test) would take 6-12 weeks depending on the number of the cloning steps.