A new, high-throughput digital PCR technology called Droplet Digital PCR™ (ddPCR™) was shown to provide an absolute measure of nucleic acids in a sample in the November 15 issue of Analytical Chemistry. According to Ben Hindson, Ph.D., the paper's first author and a Bio-Rad Laboratories, Inc. chemist, ddPCR overcomes the high costs, limited throughput, and complicated workflows that hampered adoption of earlier commercial digital PCR systems.
Study Signals Arrival of Droplet Digital™ PCR
Bio-Rad offers the ddPCR technology with its QX100™ Droplet Digital PCR system.
"The QX100 Droplet Digital PCR system's ability to count nucleic acids molecule by molecule will allow researchers to study biological systems with unprecedented precision," said Hindson. "We anticipate this will enable researchers to explore complex genetic landscapes, discover and validate new disease associations, and define a new era of molecular diagnostics."
Rise of Third-Generation PCR
First generation PCR users obtain qualitative results by end-point analysis using gel electrophoresis. Real-time PCR led to a second generation that enables relative quantification by monitoring amplification using a fluorescence chemistry added to the reaction. Quantification requires the use of a standard curve or reference gene when performing gene expression analysis. Typically, only two-fold differences in starting concentration can be observed.
Droplet Digital PCR is third-generation PCR, which enables precise measurements of DNA concentration, to provide absolute quantitation as a digital readout. The droplet technology enables digital PCR in a low-cost and practical format.
The QX100 Droplet Digital PCR system partitions the sample into ~20,000 individual droplets wherein PCR is carried out. These droplets are streamed in single file past a detector to count the negative and positive reactions. This digitization of PCR reactions allows a more reliable and sensitive measurement of nucleic acid amounts.
"The massive partitioning afforded by our Droplet Digital PCR provides orders of magnitude more precision and sensitivity than other technologies," said Hindson.
Counting Copies, Detecting Rare Alleles, and Quantifying Plasma DNA
The Analytical Chemistry paper demonstrates the utility of the QX100 Droplet Digital PCR system in three application areas of increasing interest to researchers: copy number variation (CNV) determination, rare allele detection, and plasma DNA quantification.
For CNVs, the large number of droplet partitions provides sufficient precision to accurately measure copy number states. Using the QX100 Droplet Digital PCR system to screen HapMap samples for CNVs, copy number states were shown to be completely resolved.
For the detection of rare alleles, partitioning the target mutant DNA away from highly homologous wildtype DNA increases sensitivity. With the QX100 Droplet Digital PCR system, researchers detected a 0.001% mutant fraction of BRAF V600E, which is 1,000 times more sensitive than real-time PCR.
Finally, Droplet Digital PCR enabled accurate quantitation of highly fragmented cell-free DNA in maternal plasma, an important sample matrix for emerging noninvasive tests.
For more information on the QX100 Droplet Digital PCR system, visit the company article page.