In a recent BioTechniques podcast, Drs. Matthew Strain and Daniel Kuritzkes talk about how droplet digital PCR was used to verify that an baby born with HIV had indeed been cured. Listen to the podcast below:
Posts Tagged ‘droplet digital pcr’
Professor Alec Morley, a pioneer in digital PCR, has chosen Bio-Rad Laboratories’ QX100 Droplet Digital PCR system to develop a diagnostic test for chronic myeloid leukemia (CML).
In 1992, Prof. Morley and his lab at Flinders University and Medical Center in Adelaide, South Australia, published a general method called “limiting dilution PCR” for quantifying PCR targets. As a proof of concept, they used this method for the quantification of marker mutations in acute leukemia. By diluting DNA samples so that only one or two copies per well were present and then amplifying those copies with PCR, Morley’s team was able to detect two copies of leukemic DNA against a background of 160,000 normal genomes.
They subsequently reported in The Lancet that the outcome of acute leukemia can be predicted by measuring the response to treatment using limiting dilution PCR to quantify the leukemic cells at high sensitivity. In later work, the Morley Lab used real-time quantitative PCR (qPCR) to develop a highly sensitive method for isolating and quantifying the chromosomal translocation that is typically associated with CML.
Using droplet digital PCR to diagnose leukemia
Because the translocation point for each patient is different in CML, real-time PCR conditions may vary from patient to patient and may therefore produce different results. The lab has now returned to digital PCR.
“Advancements in digital PCR have given us the ability to overcome variations in real-time PCR amplification efficiency and have also enabled us to do away with using a standard curve,” Prof. Morley said.
Monoquant, a company associated with Flinders University, recently used Bio-Rad’s QX100 system to refine the new clinical test for CML. Not only does the instrument offer high sensitivity, it also removes variability in amplification efficiency that results from using patient-specific PCR primers, a traditional sticking point for the FDA. Monoquant hopes the results from the QX100 system will fast-track the FDA approval process for its test.
“It’s a great feeling knowing that something we helped create is propelling our work today,” Prof. Morley said. “We are hoping that this new test we’re developing will offer a better degree of monitoring and better disease management for patients by tracking the progression or remission of CML.”
Independent Study Validates Bio Rad’s Droplet Digital™ PCR Technology for Analyzing Archival Cancer Samples:: Posted by American Biotechnologist on 12-13-2012
The Ji Research Group at Stanford University, in collaboration with Bio-Rad’s Digital Biology Center, demonstrated that the QX100™ Droplet Digital PCR (ddPCR™) system enables accurate and precise measurements of cancer genome amplifications in archival cancer tissue samples. The results of their study were published online in Translational Medicine.
“The cancer research community is greatly interested in accurately identifying and characterizing genome amplifications and other copy number variations because they are a critical component for understanding and treating human cancers,” said Dr. Hanlee Ji, MD, director of the Ji Research Group. “Using ddPCR, we demonstrated the superiority of this method for copy number analysis of DNA in archival material.”
Certain copy number variations (CNVs) known as genomic amplifications may lead to overexpression of specific oncogenes that drive cancer development. Targeting amplified oncogenes could move us closer to long-sought personalized therapies for cancer treatment.
Detecting amplifications in cancer tissue is technically challenging for two reasons. One is that normal tissue is known to dilute the presence of genomic amplifications. The other is that clinical samples are typically of poor quality because they are traditionally processed as formalin-fixed, paraffin-embedded (FFPE) tissues. This preservation method leads to irreversible damage to the genomic DNA. More sensitive methods of evaluation are thus needed to overcome the poor DNA quality found in FFPE samples and to detect small fractions of tumor DNA.
Bio-Rad’s QX100 system partitions samples into 20,000 droplets for individual PCR reactions. This partitioning reduces background interference and provides a more reliable measurement of a target sequence within a complex sample. As a result, measurement precision is less affected by suboptimal PCR conditions in FFPE samples than when less sensitive methods are used.
Dr. Ji and Digital Biology Center researchers tested the QX100 system to determine its effectiveness in detecting cancer gene amplifications in FFPE cancer tissue samples. They diluted gastric cancer genomic DNA containing an FGFR2 gene amplification in decreasing ratios with DNA from a normal genome sample. Their analysis confirmed the accuracy, reproducibility, and sensitivity of ddPCR in quantifying the FGFR2-amplified gene even when there was a 1,000-fold dilution with normal genome DNA.
The researchers then compared qPCR and ddPCR methods for measuring FGFR2 gene amplifications. They confirmed the presence of an approximately sevenfold amplification of the FGFR2 locus in an FFPE-processed gastric tumor using ddPCR. That amplification was very similar to the value determined using a microarray analysis on a “matched” flash-frozen sample. In contrast, qPCR analysis of the same FFPE tumor sample found a copy number estimate of 35, demonstrating that ddPCR is more accurate than qPCR for determining copy number variants in these FFPE-derived samples.
“We were able to demonstrate that ddPCR provides the sensitivity needed to detect genomic amplifications in archival material,” said Dr. Ji. “Now we can conduct a variety of genomic studies using the QX100 system that could not have been done using traditional methods, such as real-time PCR.”
For further details about the QX100, please visit http://bit.ly/ddPCR_QX100.
Evaluating a treatment for AIDS. Analyzing archival cancer samples. Tracking the RNA of a mutated gene known to cause cancer. Researchers recently met in San Diego to discuss these and many other ways they are using Bio-Rad Laboratories, Inc.’s QX100 Droplet Digital PCR system to achieve their research goals. Bio-Rad’s second QX100 User Group Meeting will be held Dec. 6–7, 2012 in Boston.
“Our first user group meeting demonstrated substantial traction for droplet digital PCR in applications that require unprecedented levels of sensitivity, precision, and reproducibility, such as the measurement of copy number variation and rare mutation/sequence detection,” said Viresh Patel, digital PCR marketing manager at Bio-Rad.
The research community has embraced the QX100 system since its release in October, 2011, as evidenced both by industry awards and the strong sales and market growth of the instrument, which is expected to continue. A recent survey conducted by The Scientist and research firm Frost & Sullivan found that 30 percent of non-digital PCR users plan to implement digital PCR in their labs in 2013. Frost & Sullivan believes the QX100 system will lead the digital PCR marketplace due to the instrument’s simplicity, performance, and affordability.
Researchers Share Droplet Digital Success Stories
During the first QX100 User Group Meeting in San Diego, academic, industry, and government researchers spoke about the benefits of droplet digital PCR (ddPCR™). Kerry Emslie, manager of the Bioanalysis Group at Australia’s National Measurement Institute, presented research published in Analytical Chemistry evaluating the performance of the QX100 system in quantifying DNA copy numbers. Using lambda genomic DNA as a model, she concluded that the system’s results are more precise than those typically observed using either real-time PCR or other digital PCR systems based on microfluidic chambers.
Dr. Hanlee Ji of the Stanford University School of Medicine spoke about his experience analyzing archival cancer samples using ddPCR analysis. In a recent report in Translational Medicine, Dr. Ji demonstrated that ddPCR improves accuracy and precision over real-time PCR when measuring copy number variation in genomic DNA from formalin-fixed paraffin-embedded (FFPE) cancer tissue.
“We run droplet digital PCR routinely,” said Dr. Ji. “It’s part of our set of genome technologies that allow us to conduct translational studies that have clinical implications.”
Users also presented research demonstrating the ultra-sensitivity of ddPCR for detecting rare events. Highlights included a contract research organization that screens for EGFR mutations in circulating nucleic acids, biotech researchers who quantify rare HIV DNA targets, and the development of a laboratory test that tracks the transcript of a fusion gene that activates cancer.
For more information about the upcoming Boston user group meeting, please contact Viresh Patel at viresh_patel(at)bio-rad.com or 925-474-8602.
Cytogenic studies over the past 50 years have hinted at the impact that copy number variations (CNVs) can have on phenotypic traits and disease susceptibility. Given the high incidence and clinical impact of CNVs, a precise, rapid and cost-effective method is needed for high-throughput validation of candidate CNV associations and for subsequent routing deployment in diagnostic settings. The predominant method used to validate CNVs in larger population is real-time or quantitative PCR (qPCR), which measures the relative rates of fluorescence increases during the exponential amplification of target and single-copy reference genes. The accuracy and precision of these measurements can be impacted by multiple factors including differences in amplification rates between the target and reference genes, variations in their amplification rates during qPCR, sampling error due to DNA concentration and analysis errors. Weaver et al. rigorously characterized these factors and found that systemic errors can be addressed by increasing the number of replicates to achieve the desired precision. however, the required number of replicates increases rapidly as finer discrimination is desired, with four replicates required to distinguish a twofold difference and up to 18 replicates to distinguish a 1.25-fold difference.
Read Digital PCR – Probing Copy Number Variations Using Bio-Rad’s QX100 Droplet Digital PCR System to learn more on how droplet digital PCR (ddPCR) can be used to determine small fold differences for higher-order CNV states.