Archive for the ‘Droplet Digital PCR’ Category
Extremely Rare Mitochondrial DNA Deletions Associated with Aging Can Be Accurately Detected with Droplet Digital™ PCR:: Posted by American Biotechnologist on 09-12-2013
A 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.
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.
Bio-Rad Laboratories, Inc. announced the launch of its PrimePCR™ assays for Droplet Digital™ PCR. These new predesigned assays for mutation detection and copy number variation (CNV) have been experimentally validated to provide single-copy PCR resolution without a standard curve.
Droplet Digital PCR (ddPCR™) technology provides an absolute measure of target DNA molecules. Researchers can use ddPCR assays to accelerate discovery and enable new research strategies for inherited disorders, cancer, and infectious diseases.
“We are excited because these are the first assays that have been made available as fully validated for digital PCR,” said Richard Kurtz, marketing manager of the Gene Expression Division at Bio-Rad. “These predesigned assays remove the burden of assay design and make digital PCR even easier for our customers.”
In this first release, Bio-Rad offers its customers 14 mutation detection assays with matching wild-type assays for biologically relevant and highly prevalent cancer mutation targets chosen from the COSMIC database. Commonly studied cancer gene mutations such as BRAF V600E and EGFR T790M are among the first set of assays available.
The assays, combined with the sensitivity of Bio-Rad’s ddPCR system, are capable of detecting a single mutant copy in a background of 2,000 or more wild-type molecules (0.05% mutation frequency). The precision of ddPCR also enables cancer researchers to discriminate small-fold copy number changes with the 62 assays targeting common cancer genes and two reference target assays now available.
Bio-Rad has partnered with Biogazelle, a qPCR data analysis and services company, and Integrated DNA Technologies (IDT), a world-class manufacturer of oligonucleotides, to design, optimize, and experimentally validate the assays. The PrimePCR ddPCR assays employ universal cycling conditions and do not require optimization. Primer specificity has been validated by next-generation sequencing, and all assays have been validated on Bio-Rad’s QX100™ Droplet Digital PCR system.
PrimePCR ddPCR assays are available in multiple reaction sizes.
For more information on Bio-Rad’s PrimePCR products, please visit www.bio-rad.com/PrimePCR.
Researchers from the University of California, San Francisco, and Bio-Rad have demonstrated that Bio-Rad’s Droplet Digital PCR (ddPCR) technology can dramatically improve the sensitivity, precision, and throughput of a popular assay for telomerase activity.
Currently, researchers are investigating telomerase activity as a biomarker for cancer diagnosis and as a target for anticancer drugs. Measuring it’s activity more sensitivity may enhance our understanding of it’s role in diseases.
This research was presented at the American Association of Cancer Research (AACR) Annual Meeting on Tuesday, April 9.
You can view the press release here: http://bit.ly/12KJyUk