DNA sequencing to detect genetic mutations can aid in the diagnosis and selection of treatment for cancer. Current methods of testing DNA samples, Sanger sequencing and pyrosequencing, occasionally produce complex results that can be difficult or impossible to interpret. Scientists at the Johns Hopkins University School of Medicine have developed a free software program, Pyromaker, that can more accurately identify such complex genetic mutations.
Pyromaker is a web-based application that produces simulated pyrograms based on user input including the percentage of tumor and normal cells, the wild-type sequence, the dispensation order, and any number of mutant sequences. Pyromaker calculates the relative mutant and wild-type allele percentages and then uses these to generate the expected signal at each point in the dispensation sequence. The final result is a virtual trace of the expected pyrogram.
The researchers validated Pyromaker against actual pyrograms containing common mutations in the KRAS gene, which plays an important role in the pathogenesis of a variety of tumors. The actual pyrograms and virtual pyrograms were quantitatively identical for all mutations tested.
They then demonstrated that all codon 12 and 13 single and complex mutations generate unique pyrograms. However, some complex mutations were indistinguishable from single base mutations, indicating that complex mutations may be underreported. Working with two complex pyrograms that were difficult to interpret initially, the researchers identified five approaches to resolve them: Sanger sequencing alone, hypothesis testing with Pyromaker, Pyromaker iterative mutation re-creation, melting curve analysis, and TA cloning with Sanger sequencing.
Senior author James R. Eshleman, MD, PhD, Professor of Pathology and Oncology, Associate Director, Molecular Diagnostics Laboratory, Johns Hopkins University School of Medicine, explains, "User-directed hypothesis testing allows for generating virtual traces that can be compared to the actual data to clarify ambiguous results from pyrosequencing and the Sanger method. Alternatively, Pyromaker can quickly and efficiently test the possibilities that can explain a complicated polysequencing result." Both strategies were able to successfully identify the complex mutations.
TA cloning and sequencing also provided unequivocal interpretation, but this method is labor intensive, risks plasmid contamination of the laboratory, may delay reporting, and is not routinely used in most clinical diagnostic laboratories.
"Although pyrosequencing and Sanger sequencing are both powerful tools to resolve most mutations, for certain complex cases, neither of them alone is enough to provide a definitive interpretation," notes Dr. Eshleman. "Additional methods, such as Pyromaker analysis or TA cloning and sequencing, allow one to definitively diagnose the variant allele. Pyromaker is available free online and can be accessed from any computer with internet access. Iterative Pyromaker analysis is the least expensive and fastest method to resolve these cases."
Pyromaker has been made freely available at http://pyromaker.pathology.jhmi.edu.
The article is "A Virtual Pyrogram Generator to Resolve Complex Pyrosequencing Results," by G. Chen, M.T. Olson, A. O'Neill, A. Norris-Kirby, K. Beierl, S. Harada, M. Debeljak, K. Rivera-Roman, S. Finley, A. Stafford, C.D. Gocke, M-T. Lin, and J. R. Eshleman (doi: 10.1016/j.jmoldx.2011.12.001). The Journal of Molecular Diagnostics, Volume 14, Issue 2 (March 2012) published by Elsevier.
David Sampson | EurekAlert!
Human skin is an important source of ammonia emissions
27.05.2020 | Max-Planck-Institut für Chemie
Biotechnology: Triggered by light, a novel way to switch on an enzyme
27.05.2020 | Westfälische Wilhelms-Universität Münster
In living cells, enzymes drive biochemical metabolic processes enabling reactions to take place efficiently. It is this very ability which allows them to be used as catalysts in biotechnology, for example to create chemical products such as pharmaceutics. Researchers now identified an enzyme that, when illuminated with blue light, becomes catalytically active and initiates a reaction that was previously unknown in enzymatics. The study was published in "Nature Communications".
Enzymes: they are the central drivers for biochemical metabolic processes in every living cell, enabling reactions to take place efficiently. It is this very...
Early detection of tumors is extremely important in treating cancer. A new technique developed by researchers at the University of California, Davis offers a significant advance in using magnetic resonance imaging to pick out even very small tumors from normal tissue. The work is published May 25 in the journal Nature Nanotechnology.
researchers at the University of California, Davis offers a significant advance in using magnetic resonance imaging to pick out even very small tumors from...
Microelectronics as a key technology enables numerous innovations in the field of intelligent medical technology. The Fraunhofer Institute for Biomedical Engineering IBMT coordinates the BMBF cooperative project "I-call" realizing the first electronic system for ultrasound-based, safe and interference-resistant data transmission between implants in the human body.
When microelectronic systems are used for medical applications, they have to meet high requirements in terms of biocompatibility, reliability, energy...
Thomas Heine, Professor of Theoretical Chemistry at TU Dresden, together with his team, first predicted a topological 2D polymer in 2019. Only one year later, an international team led by Italian researchers was able to synthesize these materials and experimentally prove their topological properties. For the renowned journal Nature Materials, this was the occasion to invite Thomas Heine to a News and Views article, which was published this week. Under the title "Making 2D Topological Polymers a reality" Prof. Heine describes how his theory became a reality.
Ultrathin materials are extremely interesting as building blocks for next generation nano electronic devices, as it is much easier to make circuits and other...
Scientists took a leukocyte as the blueprint and developed a microrobot that has the size, shape and moving capabilities of a white blood cell. Simulating a blood vessel in a laboratory setting, they succeeded in magnetically navigating the ball-shaped microroller through this dynamic and dense environment. The drug-delivery vehicle withstood the simulated blood flow, pushing the developments in targeted drug delivery a step further: inside the body, there is no better access route to all tissues and organs than the circulatory system. A robot that could actually travel through this finely woven web would revolutionize the minimally-invasive treatment of illnesses.
A team of scientists from the Max Planck Institute for Intelligent Systems (MPI-IS) in Stuttgart invented a tiny microrobot that resembles a white blood cell...
19.05.2020 | Event News
07.04.2020 | Event News
06.04.2020 | Event News
27.05.2020 | Information Technology
27.05.2020 | Physics and Astronomy
27.05.2020 | Earth Sciences