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Archive for the ‘Droplet Digital PCR’ Category

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.

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.

Breaking Leukemia’s Limits of Detection with Droplet Digital™ PCR

 :: Posted by American Biotechnologist on 09-17-2013

Extremely Rare Mitochondrial DNA Deletions Associated with Aging Can Be Accurately Detected with Droplet Digital™ PCR

 :: Posted by American Biotechnologist on 09-12-2013

jason bielasA study published recently in Aging Cell identifies a new tool to accurately analyze extremely rare mitochondrial DNA (mtDNA) deletions associated with a range of diseases and disorders as well as aging. This approach, which relies on Droplet Digital PCR (ddPCR™) technology, will help researchers explore mtDNA deletions as potential disease biomarkers.

The accumulation of mtDNA mutations is associated with aging, neuromuscular disorders, and cancer. However, methods to probe the underlying mechanisms behind this mutagenesis have been limited by their inability to accurately quantify and characterize new deletion events, which may occur at a frequency as low as one deletion event per 100 million mitochondrial genomes in normal tissue. To address these limitations, researchers at the Seattle, Washington–based Fred Hutchinson Cancer Research Center developed a ddPCR-based assay known as “Digital Deletion Detection” (3D) that allows for the high-resolution analysis of these rare deletions.

“It is incredibly difficult to study mtDNA mutations, let alone deletions, within the genome,” said Dr. Jason Bielas, assistant member of the Public Health Sciences Division at Fred Hutchinson Cancer Research Center and lead author of the study. “Our 3D assay shows significant improvement in specificity, sensitivity, and accuracy over conventional methods such as those that rely on real-time PCR.”

Bielas added, “The increase in throughput afforded by Droplet Digital PCR shortened the analysis of deletion events to days compared to months using previous digital PCR methods. Without the technology, we could not have made this discovery.”

At the center of the study was Bio-Rad Laboratories’ QX100™ ddPCR system. Using the QX100 system, Bielas and his team analyzed eight billion human brain mtDNA genomes and identified more than 100,000 genomes with a deletion. They discovered that, contrary to popular belief, the majority of the increase in mtDNA deletions was not caused by new deletions but rather by the expansion of previous deletions. They hypothesized that the expansion of existing mutations should be considered the primary factor contributing to age-related accumulation of mtDNA deletions.

How the 3D Assay Works
3D is a novel three-step process that includes enrichment for deletion-bearing molecules, single-molecule partitioning of genomes into droplets for direct quantification via ddPCR, and breakpoint characterization using next-generation sequencing.

Once the enrichment process is completed using methods previously developed by Bielas and colleagues, the concentration of molecules within the droplets is adjusted with the QX100 system so that the majority of droplets contain no mutant genomes, while a small fraction contain only one. This process allows each deletion to be amplified without bias and without introducing the artifacts that are common in qPCR.

Following amplification, deletions can be analyzed using ddPCR to determine the absolute concentration of mutated molecules. Using the relationship between droplet fluorescence and amplicon size, Bielas and his team were able to characterize the size and complexity (whether they were a result of a few clonal expansions or a large collection of random deletions) of rare mitochondrial deletions in human brain samples.

The 3D assay provides an important new tool that will allow researchers to better study the mechanisms of deletion formation and expansion, and their role in aging. Droplet Digital PCR’s high throughput and increased sensitivity will also allow Bielas’ lab to target other low-level disease-causing mtDNA deletions in skeletal muscle, brain tissue, and blood.

Droplet Digital™ PCR provides accurate quantification of next-generation sequencing libraries

 :: Posted by American Biotechnologist on 08-19-2013

A study published today found that Droplet Digital PCR (ddPCR™) can be used as an accurate and precise method for quality control of next-generation sequencing (NGS) libraries. NGS library QC is essential to optimizing sequencing data yield, thereby increasing efficiency and throughput while lowering cost. The research was published in the in the August issue of Biotechniques.

“While real-time PCR has traditionally been used to quantify libraries, we determined that the only truly accurate way to reproducibly quantify our NGS libraries is with ddPCR,” said Dr. Jason Bielas, lead author and Assistant Member in the Public Health Sciences Division at Fred Hutchinson Cancer Research Center in Seattle, Wash.

Quantifying NGS Libraries and Why It Matters

Various commercial NGS technologies require users to load a precise number of viable DNA library molecules onto the instrument to optimize data yield. Performing a sequencing run with either too many or too few library molecules results in compromised data and sometimes no data at all – wasting sample, expensive reagents, user time, and instrument time.

Moreover, fewer bases might be sequenced if library molecules are not the appropriate length to fully utilize the sequencing platform, thus limiting throughput. Given this, quantifying library molecules and determining fragment size range have become crucial steps in library preparation.

NGS instrument manufacturers recommend quantifying libraries using real-time quantitative PCR (qPCR) and determining their size range using gel or capillary electrophoresis. Each of these has its limitations, though, and the steps recommended to address them, can be time-consuming and expensive.

Advantages of ddPCR for Quantifying NGS Libraries

To simultaneously quantify and determine the size distribution of target DNA with a single ddPCR assay, Dr. Bielas and his team exploited a relationship between droplet fluorescence and amplicon size. They confirmed the accuracy and precision of this method by applying it to NGS library preparation.

The ddPCR assay they designed – known as QuantiSize – was developed using the QX100 ddPCR system from Bio-Rad Laboratories. QuantiSize offers the ability to determine the absolute quantity and the detailed size distribution of target DNA in a single ddPCR reaction well, thus avoiding the drawbacks of other independent quantification and size determination methods.

“Now that we have discovered this new correlation, we can also use ddPCR to extract more information on the characteristics of DNA based on the range of fluorescence that can occur within each droplet,” said Bielas.

Having demonstrated the efficacy of this technique, Dr. Bielas is now planning to leverage the relationship between ddPCR fluorescence and amplicon size to explore mutagenic deletion events in both the human nuclear and mitochondrial genomes.