ThermoSleuth

ThermoSleuth™ is a PCR assay design tool developed by DNA Software that scans multiple primers against collections of genomes to identify all hybridization and mishybridization sites, including false amplicons. It is intended for researchers working on DNA diagnostic assays who need to verify primer specificity and eliminate false positives caused by off-target primer binding.

Unlike BLAST, which scores hits based on sequence similarity, ThermoSleuth applies thermodynamic models to evaluate primer and probe hybridization. It operates through a cloud-based infrastructure, giving researchers access to the computational capacity needed to work with large genome databases without requiring local storage or processing resources.

Core Capabilities for Primer Specificity

• Scans oligos against large genome databases, including the human genome and microbiome, using BLAST speed combined with thermodynamic scoring.
• Applies proper thermodynamic scoring that differentiates GC pairs from AT pairs, unlike standard BLAST.
• Scores hits based on complementarity rather than similarity.
• Detects all false amplicons resulting from primer mishybridization.
• Accounts for 3' extensibility and stable mismatches when evaluating hits.
• Allows configuration of solution conditions including salt concentration, buffers, additives, and other experimental factors.
• Captures up to 50 times as many thermodynamically stable and extensible hits compared to BLAST.

Genome Database and Playlist Features

• Includes a repository of curated and pre-formatted sequence databases.
• Supports single genome scans, multiple genome scans, and user-defined genome playlists.
• Allows users to build background databases of any size without restrictions on memory, storage, or computational capacity.
• Enables sharing of target sequences and analysis results between users.

Feature Comparison Highlights

• Supports specificity checking and sensitivity checking.
• Detects extensible hits and probe hybridization.
• Evaluates probe dyes and minor groove binders.
• Correctly scores mismatches by identity, bulges, and target dangling ends.
• Detects amplicons and allows download of amplicons plus tails.
• Provides adjustable primer extensibility settings.
• Offers a graphic overview of results and a genome view with detailed thermodynamics.
• Supports multiplex capability.
• Differentiates DNA from RNA backbones.
• Supports modified backbones and modified bases.
• Simulates conditions using monovalent and divalent salt, different temperatures, and different primer concentrations.

Licensing Packages

• Base Package: Each licensed user can access ThermoSleuth through a secure web UI portal, analyze hybridization of up to 500 query oligos per job, query target sequences of up to 10 billion nucleotides in total length per job, execute up to 2 concurrent analysis jobs, run up to 100 hours of hosted hybridization analysis with unlimited results storage, and share target sequences and results with other users.
• ThermoSleuth Modifieds Package: Adds the ability to accept and evaluate input query sequences containing modified nucleotides, and provides advanced analysis using empirically derived hybridization parameters and pairing models for modified nucleotides.

ThermoSleuth is delivered as a cloud-hosted platform accessible via a web UI, removing the need for local computational infrastructure. A free trial and scheduled demos are available for organizations evaluating the tool.