Digital PCR (dPCR) is the latest technology in the PCR arsenal. The driving innovation behind dPCR is sample partitioning. Each partition—via microfluidic chips, channels or droplets—serves as an individual PCR reaction, delivering remarkable sensitivity and quantification of target nucleic acids. As actual molecules are measured, the necessity of establishing a standard curve—a primary component of real-time, or quantitative, PCR (qPCR)—is eliminated. Digital PCR is not always necessary, however. The choice to go digital depends largely on your application and the level of sensitivity required. Below, we offer some guidelines for when dPCR is the best fit for your application.
When you need to detect rare events
One application particularly well-suited for dPCR is rare event detection in clinical studies. Biocompare spoke with Gary Lee, senior scientist at Sangamo, Inc. regarding the company’s adoption of dPCR in late 2011. Sangamo specializes in gene therapy, engineering DNA-binding zinc finger protein (ZFP) nucleases that can tailor gene expression. One such therapy, currently in two separate phase II clinical trials, may someday provide a functional cure for HIV, offering an alternative to conventional drug treatment.
“If you’re using traditional applications where real-time PCR gives you precise answers, there’s no need to go digital,” says Lee. “Because we’re dealing with a low copy event, we couldn’t get readings at all in some cases using qPCR. Digital PCR is essential for us to track the progress of a patient’s therapy.”
The presence of HIV DNA in infected patients’ blood samples is a rare event, often occurring in only 1/1,000 to 1/10,000 specialized immune cells in an infected person’s genome. Sangamo’s efforts to develop an assay that could measure any substantial change in a patient’s CD4+ T cells (the specialized immune cells) using qPCR were unsuccessful. The presence of significant background genomic DNA (gDNA) inhibits amplification of low copy numbers of HIV target sequence. The partitioning technology of dPCR greatly improves the ratio of positive to negative template and thus the efficiency of amplification to the point where Sangamo could detect about 100 copies of HIV sequence per million cells, in some samples.
When absolute (versus relative) quantification is needed
qPCR measures the number of copies in a sample relative to a serial diluted standard curve. The measurement is relative to the sample and not the actual number of copies in a sample itself (dPCR's ‘absolute quantification’). There is a heavy reliance, then, on having an accurate standard curve. It can also take considerable time to set up a DNA plasmid stock when establishing a standard. For instance, if you want to investigate a novel or unique recombinant DNA sequence, you must first derive the genetic sequence, clone the sequence into a plasmid, generate a plasmid stock, linearize the plasmid and then test your primer probes. Digital PCR saves start-up time by eliminating the requirement of generating a standard.
When you need to detect fine variations, as in copy number analysis
Digital PCR is also especially useful for applications like rare allele detection, validating next generation sequencing experiments and determining fine copy number variances. For instance, some genes in the human genome have a certain number of repeats or specific mutations that a researcher may want to detect in a specific population to understand influences on gene expression or the risk of disease. In some cases, there are as many as five copy number variations. To study this effectively, you must be able to measure the copy number of a genomic sequence variation with great precision in multiple samples. Real-time PCR would require running numerous replicates to detect such fine variances.
Sometimes qPCR fits the bill
It was only a decade ago that qPCR set the standard for sensitive DNA quantification. Sangamo uses both qPCR and dPCR at their facility, and for most applications qPCR performs as expected. As Lee points out, both systems are set up to measure the same physical DNA copy number in a sample. Although dPCR measures the absolute copy number directly and qPCR relies on extrapolation to a standard, there shouldn’t be any difference in the measurements using qPCR or dPCR until you run up against the limits of detection with qPCR, i.e., when exploring lower target levels to background gDNA. When qPCR is performed correctly, results are equally reproducible. Although dPCR is more precise and robust for certain applications, it can be more costly per reaction and there are some high throughput limitations that manufacturers are quickly working to fill. With dPCR sales expected to climb by 30% in 2013, third generation PCR is likely to expand into other applications, as well.
In closing, Lee adds, “The need for dPCR is really application dependent. qPCR is so widely used you can get a lot of support and there are many reagents to choose from. It’s when you’re facing certain limitations that dPCR can really benefit your research.”