Dedicated to anyone who has ever been scooped. What a dreadful feeling! Hope this cheers you up.
One of biology’s most fundamental processes is something called transcription. It is just one step of many required to build proteins—and without it life would not exist. However, many aspects of transcription remain shrouded in mystery. But now, scientists at the Gladstone Institutes are shedding light on key aspects of transcription, and in so doing are coming even closer to understanding the importance of this process in the growth and development of cells—as well as what happens when this process goes awry.
In the latest issue of Molecular Cell, researchers in the laboratory of Gladstone Investigator Melanie Ott, MD, PhD, describe the intriguing behavior of a protein called RNA polymerase II (RNAPII). The RNAPII protein is an enzyme, a catalyst that guides the transcription process by copying DNA into RNA, which forms a disposable blueprint for making proteins. Scientists have long known that RNAPII appears to stall or “pause” at specific genes early in transcription. But they were not sure as why.
Since its introduction in 2011, Bio-Rad Laboratory’s Droplet Digital PCR (ddPCR™) technology has demonstrated the potential to be a transformative technology, particularly in clinical applications.
In the past, tools developed for such applications have been limited by their inadequate precision and/or their lack of sensitivity for detecting rare species. But thanks to ddPCR technology, researchers can now focus on more of these “needle-in-a-haystack” problems. Less than two years since Bio-Rad brought ddPCR systems to the market, their application has resulted in nearly 50 peer-reviewed publications citing the technology.
The advantages of ddPCR technology have already had an important impact on medical research.
“The HIV community, for instance, has benefited from ddPCR’s ability to make more sensitive measurements, which can also depend on its attribute of increased precision,” said George Karlin-Neumann, the scientific affairs director at Bio-Rad’s Digital Biology Center. “To have greater sensitivity down to very, very low levels depends on being able to distinguish something from nothing. You need a system that inherently has very low noise.”
Droplet Digital PCR Is a Sensitive Tool for Detecting Residual HIV DNA
A great example of the clinical potential of ddPCR systems is the work of Matt Strain and Douglas Richman of the Center for AIDS Research at the University of California, San Diego School of Medicine, who validated the technology’s performance in HIV provirus detection. Subsequently, in collaboration with Deborah Persaud of Johns Hopkins Children’s Medical Center in Baltimore, the researchers used the technology to demonstrate that an infant born with HIV was functionally cured.
In a recent BioTechniques podcast, Dr. Strain said that ddPCR assays demonstrate an increase in precision and accuracy over their entire dynamic range relative to real-time PCR assays, particularly at low numbers. Additionally, the total cost per sample of Droplet Digital PCR assays is at least 10 to 100 times less than that of older chip-based digital PCR systems.
“When you’re talking about factors of a hundred or more in cost, there really isn’t any comparison,” said Dr. Strain.
Dr. Richman will present information on using ddPCR to detect latent HIV, including assaying rare events in a large number of cells and retrieving clinically relevant data.
A Glimpse at Droplet Digital PCR’s Future in Diagnostics
The research group headed by Hanlee Ji, an assistant professor at Stanford University School of Medicine, focuses on translational and clinical questions of cancer genetics that, once answered, have the potential to improve cancer patient care. The investigators have developed numerous methods for the accurate interrogation of cancer genomes that overcome challenges associated with clinical samples and the genetic variability resulting from tumor evolution. In this endeavor, Droplet Digital PCR is one of their chief tools.
“Droplet Digital PCR has accelerated our discoveries,” said Dr. Ji. “Given its ease of use, superior performance in terms of accuracy, and rapid development time for novel assays, Droplet Digital PCR has repeatedly demonstrated its vast utility and potential for future diagnostic application.”
Dr. Ji recently gave a talk on using ddPCR technology to track the presence, expansion, and disappearance of pathogenic genetic variants in cancer, infectious diseases, and other human diseases over time, and also discussed the technology’s potential for highly informative diagnostics. He now uses Bio-Rad’s recently launched second-generation ddPCR instrument, the QX200™ Droplet Digital PCR system, the only digital PCR system that works with both DNA-binding dye and TaqMan probe chemistries.
The QX100™ system boasts a lineup of prestigious users including:
- Jim Huggett,the author of the digital MIQE (dMIQE) guidelines and a scientist at LGC (the UK’s designated National Measurement Institute for chemical and bioanalytical measurement)
- David Dodd of the University of Texas, Southwestern
- Leonardo Pinheiro of Australia’s National Measurement Institute
- Ross Haynes of the U.S. National Institute of Standards and Technology
- Vicki Hwang of the University of California, Davis
- Alec Morley of Flinders University and co-author of the first paper to use digital PCR
- Keith Jerome of the University of Washington
- Gary Lee of Sangamo BioSciences
- Sabita Sankar of MolecularMD
- Donna Sullivan of the University of Mississippi Medical Center
There are also a number of emerging applications using ddPCR technology including microRNAs, single-cell gene expression, gene linkage, multiplexing, EvaGreen applications, and validating next-generation sequencing data.
For more information on the QX200 Droplet Digital PCR system, visit www.bio-rad.com/QX200.
To view Bio-Rad’s six-part webinar series on Droplet Digital PCR and the complete list of Droplet Digital PCR system publications, visit http://www.bio-rad.com/ddPCR-Webinars.
The following videos help explain:
1. What is the Nogoya Protocol
2. Why is the Nogoya Protocol necessary?
The United Nations (UN) is working to ensure that the benefits of genetic resources are shared in a fair and equitable way via the Nagoya Protocol to the Convention on Biological Diversity.
The Nagoya Protocol was adopted in 2010 to provide a transparent legal framework for sharing genetic resources. “Its objective is the fair and equitable sharing of benefits arising from the utilization of genetic resources, thereby contributing to the conservation and sustainable use of biodiversity,” according to the UN.
A new report from the Synthetic Biology Project at the Woodrow Wilson International Center for Scholars looks at how the protocol may affect U.S. researchers working in the field of synthetic biology.