Solving Primer Specificity

The problem of false positives in PCR

The most common cause of false positives in PCR is the formation of false amplicons due to primer mishybridization which is not detected by BLAST because it scores hits incorrectly using sequence similarity rather than using the proper rules for match and mismatch complementarity.   Additionally, many researchers do not have access to the computational capacity or storage required of modern genomic-based assay designs.

The solution to false positives in PCR

ThermoBLAST CE and ThermoBLAST solves the problem of primer and probe mishybridization by scanning against whole genome databases such as the human genome or microbiome using the speed of BLAST while applying the proper thermodynamic model to mishybridization and crosshybridization results.   The cloud integration of ThermoBLAST CE enables all researchers with the computational power to capture 50X as many thermodynamically stable and extensible hits as BLAST.

DNA Software introduces the full commercial release of ThermoBLAST Cloud Edition (TB-CE).

ThermoBLAST Cloud Edition Overview

ThermoBLAST Cloud Edition (TB-CE) provides a new standard for evaluating the target specificity of oligonucleotides during PCR primer or probe design. Everybody is familiar with the capabilities of NCBI BLAST, but there are no thermodynamic results and lots of unwanted and meaningless data. ThermoBLAST CE scans oligonucleotides against genomic databases, which can be organized into “playlists”, to thoroughly search for hybridization sites that can cause false positives in multiplexed diagnostic tests or hybridization-based therapeutics. The output data is then organized by thermodynamic stability, which can then be sorted according to annealing temperature to weed out those reactions that are not likely to happen under your specified temperature and salt conditions. Fast and accurate thermodynamic analysis allows for faster design and more success in the laboratory.

ThermoBLAST CE includes the following features:

  • Overcomes NCBI BLAST limitations.
  • Automatic detection of all thermodynamically stable hybridizations against huge genome databases.
  • Automatic detection of PCR amplicons for all combinations of multiplex primers against every GenBank accession in the playlist.
  • Increased speed and database management using the computational capacity of Cloud computing.
  • Huge repository of formatted and curated sequence databases.
  • Create and format custom sequence playlists in minutes.
  • Visualize hits in a new Genome viewer.

Archive past results in your secure personal account on the Cloud

The Cost of Using BLAST

Trial and error primer and probe synthesis and optimization is expensive due to a few core BLAST limitations:

  • Hits are scored on sequence similarity rather than thermodynamic affinity.
  • Simulation is not possible under actual experimental oligonucleotide concentrations and salt conditions.
  • False-amplicons and off-target effects cannot be quantified.
  • DNA/DNA, RNA/RNA, or DNA/RNA hybrid duplexes cannot be properly scored for basepair matches or mismatch geometry.
  • Oligonucleotide secondary structure such as bulges, gaps, hairpins and dangling ends are completely ignored.
  • Modified nucleotides and backbones are completely ignored.


A sample study was performed where a designed set of primers specific to the Y chromosome from the GRCh 38 Human Genome was queried in both ThermoBLAST and BLAST to compare the number of extensible hits and the number of false amplicons that were detected. The results showed that ThermoBLAST detected 765 unique extensible hits in the Y chromosome while BLAST detected only 15.  Furthermore, ThermoBLAST detected a total of 946 extensible hits for the forward primer design, where BLAST detected only 52 extensible hits. For the reverse primer ThermoBLAST found over 98% more hits than BLAST, and likewise for the forward primer ThermoBLAST found over 94% more hits than BLAST.  Questions about this study can be directed to DNA Software, Inc.

ThermoBLAST TM = Cloud Integration + Custom Genome Playlists+Genome Viewer+The Computational Capacity To Use It

  • Increased speed and database management using the computational capacity of Cloud computing
  • Huge repository of formatted and curated sequence databases
  • Create custom sequence playlists
  • Archive past results in your secure personal account on the Cloud
  • Visualize hits in Sequence and Genome viewers

ThermoBLASTTM = BLAST speed and database capabilities + OMP Thermodynamics

  • Hits are scored based on hybridization affinity rather than sequence similarity
  • Detects 100% of the false amplicons in a PCR reaction
  • Detects off-target effects of primer and probe mis- or crosshybridization
  • Properly scores DNA, RNA, DNA-RNA hybrids and LNA modifications
  • OMP thermodynamics properly scores hybridization gaps and bulges and dangling ends
  • Optimize primer and probe designs in silico under experimental salt and temperature conditions before synthesis

World-Class Science

ThermoBLASTTM was developed with grants from the NIH and the Department of Homeland Security. The result is that ThermoBLASTTM has the highest level of algorithm development and validation, providing our customers with a state-of-the-art tool.

World-Class Customers

To account for mishybridization, ThermoBLASTTM is trusted by industry and life-science leaders world-wide: CDC, FDA, USDA, NIST, Novartis, J&J, DuPont, Luminex, Life Technologies, Cepheid, Roche, Philips, Canon US Life Science, IMDx, PrimeraDx, and NABsys.