ExonSurfer: a web-tool to design primers at exon-exon junctions.

Area of Science:

• Molecular Biology
• Genetics
• Bioinformatics

Background:

• Reverse transcription quantitative PCR (RT-qPCR) is crucial for gene expression analysis in research and diagnostics.
• Designing effective RT-qPCR primers is challenging due to stringent quality requirements, including specificity, avoidance of polymorphisms, and prevention of genomic DNA amplification.
• Existing software tools often fall short in meeting all necessary primer design criteria.

Purpose of the Study:

• To introduce ExonSurfer, a novel, user-friendly web-tool for designing high-quality RT-qPCR primers.
• To address the limitations of current software in meeting comprehensive primer design requirements.

Main Methods:

• ExonSurfer integrates target selection, specificity assessment, self-complementarity checks, and polymorphism avoidance.
• Primers are designed on exon-exon junctions to prevent genomic DNA amplification.
• Genomic alignment is performed to filter primers and identify potential interactions.

Main Results:

• ExonSurfer successfully designed primer pairs for 26 targets, assessing primer efficiency, amplicon melting temperature, and length.
• Sanger sequencing confirmed that most designed primers accurately and selectively amplified target amplicons.
• The tool effectively avoids genomic DNA amplification by utilizing exon-exon junction design.

Conclusions:

• ExonSurfer provides a comprehensive solution for end-to-end primer design, ensuring transcript-specific amplification.
• The intuitive user interface offers essential specificity and amplicon details.
• ExonSurfer is available as a web tool and command-line option with source code, aiming to simplify RT-qPCR setup for researchers.