False Positives


The Problem:

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:

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). 

TB-CE provides a new standard for evaluating the target specificity of oligonucleotides and includes the following features: 

  • 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 "Free" BLAST

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

  • BLAST hits are scored on sequence similarity rather than affinity
  • BLAST cannot evaluate whether a hit is thermodynamically stable or not under experimental conditions
  • BLAST cannot quantify false amplicons or off-target effects
  • BLAST cannot properly score DNA, RNA, or DNA / RNA hybrid basepair matches or mismatches
  • BLAST cannot properly score modified nucleotide hybridization

 

ThermoBLAST CETM > BLAST

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 CE and BLAST to compare the number of extensible hits and the number of false amplicons that were detected. The results showed that ThermoBLAST CE detected 765 unique extensible hits in the Y chromosome while BLAST detected only 15.  Furthermore, ThermoBLAST CE 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 CE found over 94% more hits than BLAST.  Questions about this study can be directed to DNA Software, Inc.

ThermoBLAST CETM = 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, thereby providing the best possible accuracy and most thorough searching.

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.

DNAS has passed my tests. I’ve recommended Visual OMP. It performs extremely well.
~ Dr. Ned Sekinger, Luminex Corporation

I have been using DNAS for a long time, at least 8 years. I want to have the best tools available and that is why we use Visual OMP.
Dr. Nancy Schoenbrunner, Roche Molecular Systems

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Thursday, 06 November 2014 22:15

DNA Software introduced the full commercial release of ThermoBLAST Cloud Edition (TB-CE). TB-CE provides a new standard for evaluating the target specificity of oligonucleotides.  Click here to learn more.  

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