Posts Tagged ‘droplet digital pcr’

Bio-Rad Launches PrimePCR™ Assays for Droplet Digital™ PCR

 :: Posted by American Biotechnologist on 12-11-2013

Bio-Rad Laboratories, Inc. announced the release of its PrimePCR assays for Droplet Digital PCR systems. This release expands Bio-Rad’s current offering by an additional 46 mutation detection assays and 323 copy number assays. In addition, the existing set of gene expression qPCR primer-only assays can now be used with the recently launched QX200™ Droplet Digital PCR system, featuring EvaGreen detection capabilities.

The new ddPCR assays are the only predesigned and fully wet-lab validated assays for digital PCR, alleviating the burden placed on researchers of design and experimental optimization and offering precision and sensitivity without a standard curve. The assays enable novel research strategies and accelerate discovery for inherited disorders, cancer, and infectious diseases.

Many methods for mutation analysis offer poor selectivity and fail to detect mutation events with abundances of less than one in 100 wild-type sequences. Bio-Rad’s ddPCR technology provides an absolute measure of target DNA molecules, and together with the PrimePCR mutation detection assays, can enable the detection of one mutant molecule in the background of 100,000 wild-type sequences (0.001%). Measuring these extremely low levels of mutation abundance can lead to dramatically more sensitive and less invasive diagnostics.

Current methods, including qPCR and next-generation sequencing, also lack the resolution needed to accurately analyze copy number variation (CNV). Due to their high precision and absolute measurement capability, ddPCR assays enable the quantitative discrimination required to resolve small-fold changes in gene copy numbers. ddPCR assays can also be used to detect subsequent changes in the expression of target genes.

PrimePCR ddPCR assays are available in multiple reaction sizes and are compatible with both the QX100 and QX200 platforms using the ddPCR supermix for probes.

For more information on Bio-Rad’s PrimePCR products, please visit www.bio-rad.com/PrimePCR.

Using Droplet Digital™ PCR to Quantify Levels of HIV Reservoirs

 :: Posted by American Biotechnologist on 12-04-2013

Emerging Applications of Bio-Rad’s Droplet Digital™ PCR Technology

 :: Posted by American Biotechnologist on 11-06-2013

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.

Bio-Rad’s Droplet Digital PCR Highlighted at ASHG Annual Meeting

 :: Posted by American Biotechnologist on 10-22-2013

More accurate and precise assessment of copy number variation (CNV), the number of copies of a particular gene that are present in a genome, may lead to improved diagnosis and therapy for cancer and other diseases including autoimmune disorders. However, current technologies to determine copy number can be inaccurate, labor intensive, or prohibitively costly, diminishing their effectiveness in clinical applications.

Using Bio-Rad Laboratories’ Droplet Digital PCR (ddPCR™) technology, researchers can rapidly, accurately, and economically determine copy number states. Groups from the University of California, Davis, and the University of Colorado School of Medicine, among others, will present promising new research results at this year’s annual meeting of the American Society of Human Genetics (ASHG), held in Boston from October 22-25, 2013.

“Since its introduction in 2011, Bio-Rad Laboratory’s ddPCR technology has demonstrated the potential to be a transformative technology based on its greater precision, reproducibility, and sensitivity than conventional approaches such as real-time PCR,” said George Karlin-Neumann, the scientific affairs director at Bio-Rad’s Digital Biology Center.

Less than two years since Bio-Rad brought digital PCR systems to the market, studies using ddPCR assays have resulted in nearly 50 peer-reviewed publications.

Using ddPCR to Improve Pediatric Care

One example of how CNV determination with ddPCR technology shows promise for clinical applications is newborn CNV screening. 22q11.2 Deletion Syndrome (22q11DS) is the most common microdeletion syndrome in humans and is associated with more than 100 different diagnostic findings including craniofacial defects, developmental delay, and autism spectrum disorders. Researchers in Dr. Flora Tassone’s lab at the University of California, Davis have developed an inexpensive, rapid, sensitive, and specific alternative to fluorescent in-situ hybridization (FISH) – the currently accepted diagnostic tool – using ddPCR technology to identify newborns with 22q11DS. At the ASHG meeting, they will demonstrate for the first time the efficacy of ddPCR in large population screening studies (Program Number 2590F).

Researchers on Dr. James Sikela’s team at the University of Colorado School of Medicine are investigating DUF1220 copy number reduction and its association with microcephaly, a neurodevelopment disorder related to pediatric brain size. They will demonstrate how ddPCR is an effective technique for determining copy numbers of highly duplicated sequences such as DUF1220 and how ddPCR may be used for similar studies in future research where array comparative genomic hybridization (aCGH) analysis or qPCR are not accurate enough (Program Number 3215F).

In addition to the research findings described above, Bio-Rad’s ddPCR technology will be showcased in 11 other presentations. For more information, visit Bio-Rad’s booth at ASHG (booth #839). Bio-Rad will also be hosting an ASHG lunch workshop on Thursday, October 24 at 12:30 PM in room 211 as well as a hospitality suite on Thursday, October 24 at 7:45 PM at the New England Aquarium, 1 Central Wharf in Boston, featuring presentations from UC Davis and Harvard Medical School researchers. Please visit booth #839 for further details.

Complex Diseases Traced to Gene Copy Numbers

 :: Posted by American Biotechnologist on 10-17-2013

Duke researchers have connected very rare and precise duplications and deletions in the human genome to their complex disease consequences by duplicating them in zebrafish.

The findings are based on detailed studies of five people missing a small fragment of their genome and suffering from a mysterious syndrome of craniofacial features, visual anomalies and developmental delays.

When those patient observations were coupled to analyses of the anatomical defects in genetically altered zebrafish embryos, the researchers were able to identify the contribution specific genes made to the pathology, demonstrating a powerful tool that can now be applied to unraveling many other complex and rare human genetic conditions.

The findings are broadly important for human genetic disorders because copy-number variants (CNVs) — fragments of the genome that are either missing or existing in extra copies — are quite common in the genome. But their precise contribution to diseases has been difficult to determine because CNVs can affect the function of many genes simultaneously.

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