Strategy for automated selection of high-fidelity splice-sensitive PCR primers. (A) The first stage involves the identification of the upstream constitutive sequence (UCS), alternatively included region (AIR), and downstream constitutive sequence (DCS) in all simple alternative splicing events types; single cassette exon, alternative 5′ and 3′ splice site, and intron retention. (B) The second stage generates primer sequences to quantify the short isoform of any given alternative splicing event. The schema in the upper right shows regions that are expanded on both sides of the junctions. Black arrows indicate the orientation of the nucleotide-by-nucleotide comparison used in steps 1 and 2. Step 1: The script identifies the stretch of matching nucleotides between short and long splicing isoforms and the 5′ end of the BSP in both orientations (BSP 5′ end similarity). Step 2: The script analyzes the sequences for the presence of mismatches in the terminal 3′ end toward the long splicing isoform. (BSP 3′ end similarity.) Step 3: Sequences identified in steps 1 and 2 are merged. Sequences with a Tm > 6°C are removed from the valid sequence list. Step 4: Sequences are sorted by mismatch number and by Tm. Step 5: An in silico 5′ end extension is performed on the best sequence until adequate nucleotide composition. Step 6: The script designs an opposing primer for the BSP yielding a short-specific primer pair (SSPP). (C) The third stage generates a primer sequence to quantify the long isoform of any given alternative splicing event. The extended space design for the long-specific primer is highlighted in red (forward) and blue (reverse).
