ExonSurfer

ExonSurfer designs high-specificity primers for qPCR, targeting exon-exon junctions to ensure transcript-specific amplification while avoiding genomic DNA amplification.

Key Features:

• Exon-exon junction targeting: Designs primers at exon-exon junctions to prevent amplification of potentially contaminating genomic DNA.
• Specificity assessment: Evaluates primer specificity to target transcripts.
• Self-complementarity verification: Checks primers for self-complementarity issues.
• Polymorphism avoidance: Filters primers to avoid polymorphic regions.
• Genomic alignment: Performs genomic alignment to filter primers and predict any genomic interactions.
• Amplicon evaluation: Assesses primer efficiency, amplicon melting temperature, and amplicon length.

Scientific Applications:

• RT-qPCR primer design: Facilitates primer design for reverse transcription quantitative PCR with intercalating dyes used in gene expression analysis.
• Transcript-specific amplification: Ensures amplification specificity for target transcripts in basic and applied research as well as diagnostics.
• Genomic DNA contamination control: Prevents false signals from genomic DNA contamination in RNA-based qPCR experiments.

Methodology:

ExonSurfer combines target selection, specificity and self-complementarity assessment, polymorphism avoidance, and genomic alignment. Primer performance was validated by designing primer pairs for 26 targets and checking primer efficiency, amplicon melting temperature and length, with confirmation of targeted amplicons by Sanger sequencing.