Scientists in laboratories around the world are using the advancements in polymerase chain reaction (PCR) technology to their advantage. New tools are extending their research capabilities, like the researchers at the Ohio State University (Columbus), who are studying potential therapies for spinal muscular atrophy, or the McCarroll lab at Harvard (Cambridge, MA), which was able to find new ways of analyzing complex genome structures and relating them to human disease. Digital PCR is one of the most powerful tools a researcher has when it comes to nucleic acid detection and quantification. As a third-generation polymerase chain reaction, it enables researchers to directly quantify nucleic acids, including DNA and RNA.
The partitioning of samples into thousands of subreactions provides digital PCR with significantly improved quantification accuracy as well as the sensitivity of mutation detection relative to quantitative PCR (qPCR) and sequencing.
With the ever-increasing accessibility of digital PCR, many laboratories are choosing to use this technology for copy number variation determination, rare event detection, gene express analysis, and, most recently, next-generation sequencing. As the results grow, so do the research possibilities.
How droplet digital PCR works
Figure 1 – Partitioning minimizes competition between target sequence and highly abundant background DNA.
Droplet digital PCR (ddPCR) provides an absolute measure of target nucleic acids molecules with extreme precision, accuracy, and sensitivity. It works by first partitioning a standard 20-μL qPCR reaction into 20,000 nanoliter-sized droplets, with target and background DNA (or RNA) randomly distributed among the droplets (see Figure 1). The DNA in these droplets is then amplified using normal PCR conditions in a conventional thermocycler.
After PCR amplification, each droplet is individually interrogated, after which it provides a positive or negative fluorescent signal that indicates whether or not the target DNA was present after partitioning and amplification. Each droplet provides an independent digital measurement. Software analyzes the resulting PCR-positive and PCR-negative droplets to provide an absolute measure of target quantity. This postamplification analysis is less prone to artifacts produced by varying PCR efficiencies often present in suboptimal reactions and in the presence of certain inhibitors.
Advantages of ddPCR
The partitioning of samples to levels above 10,000 allows for more reliable and sensitive measurement of nucleic acids than conventional methods. Droplet digital PCR provides absolute standalone quantitation results that are not dependent on standard curves or control samples. This allows for the resolution of small-fold (1.2×) differences. Additionally, calculations are simplified, since values generated are numerical and calculations can be performed using standard methods, not log transformed methods, as when using qPCR.
Sample partitioning has the added advantage of reducing multiplex assay cross-reactivity and synthetic enrichment effects when looking for rare abundance mutations. Signals generated by higher-copy templates in a pooled reaction (20 μL) often make it difficult, if not impossible, to detect a lower-abundance signal. Splitting the higher concentration template into thousands of droplets allows this signal to be accounted for while diminishing its intensity, on a per droplet basis, thus permitting the rare or lower-abundance target to become easily detectable and quantifiable.
When compared to other digital PCR systems such as those that rely on microfluidic chip technology, ddPCR’s droplet technology achieves a much higher level of partitioning, which increases precision, accuracy, and lower cost per sample.
Droplet digital PCR allows researchers to easily quantify and detect samples at levels rarely seen before and to pose questions that were previously seen as too difficult or too costly to even consider.
Emerging applications of droplet digital PCR
Due to its unique features, ddPCR has proven to be suitable for a number of emerging applications, such as copy number variation, detection of rare sequences, gene expression analysis, and next-generation sequencing. Following are examples of how scientists are exploring new frontiers in their research by using ddPCR in their laboratories.
Accurate copy number variation (CNV) determination
Copy number variations comprise simple duplications to inversions, translocations, and trisomies, and are associated with phenotypes ranging from having no effect, enhanced features, to cancers and premature death. Determining the accurate quantitation of copy number in medicine can sometimes provide information on prognosis and suggest treatment modifications.
Using traditional techniques like qPCR can prove difficult when quantifying at higher copy numbers (e.g., 4 from 5 copies). However, ddPCR enables 1.2×-fold differences to be measured and can easily discriminate between integer gene copies. It also effectively detects CNV events for both individual gene targets as well as for individual SNPs. An example of using ddPCR for CNV detection is discussed in a paper by the Steve McCarroll lab at Harvard.1
In the study, McCarroll’s lab set out to understand the human chromosome 17q21.31. This chromosome contains a megabase-long inversion polymorphism as well as many uncharacterized CNVs and markers associated with family fertility, female meiotic recombination, and neurological diseases. The inverted form of the chromosome also seems to be positively selected in Europeans.
To understand the complex patterns of this genomic structure, the McCarroll lab used ddPCR to determine integer CNV segments. The researchers designed a pair of PCR primers and a hydrolysis oligonucleotide probe to both the CNV locus and a two-copy control locus and then performed ddPCR on their samples. The absolute copy number for the CNV locus was determined by comparing droplet count measurements of the CNV locus to the two-copy control locus. Using ddPCR and other methods, the McCarroll lab was able to offer new ways of analyzing complex genome structures and relating them to human disease.
Genetic variations associated with rare events such as nucleotide mutation, alteration of copy number, and deletion or insertion of nucleotides are difficult to detect due to their dilution by the overabundance of DNA or RNA from normal cells co-collected from either tissues or bodily fluids such as blood during sample acquisition. However, early detection of rare events can make all the difference in the outcome of cancer patients, as well as lead to better monitoring and more sensitive and less invasive diagnostics.
Researchers at Ohio State University used ddPCR to study therapies for spinal muscular atrophy (SMA), which is the most common genetic cause of infant death.2
SMA is caused by a deletion or mutation of the SMN1 gene that results in a degraded transcript. Researchers used antisense oligomers to see if they could raise SMN protein levels. In doing this, they found ddPCR to have many advantages for both quantification and detection of low levels of transcript. It was also highly reliable in quantification when compared with qPCR because it did not suffer from logarithmic error.
Precise gene expression analysis
Although qPCR revolutionized the field of gene expression analysis and continues to play an important role, ddPCR enables superior analytical performance over this and other techniques. It provides absolute quantitation of expression levels, especially low-abundance nucleic acid targets (such as miRNAs), with greater sensitivity and precision. In addition, an abundance of different transcripts can easily be compared directly to one another across cells, tissues, and organisms, and across labs around the world.
A forthcoming publication from the Tewari lab at the Fred Hutchinson Cancer Research Center in Seattle, WA, describes the adaptation of ddPCR for the quantification of miRNAs, its operating characteristics, a side-by-side comparison to real-time PCR, and application to absolute quantification of circulating microRNA biomarkers from the serum of prostate cancer patients and healthy controls. Compared to the current standard approach (qPCR), ddPCR demonstrates substantially better day-to-day reproducibility of absolute quantification and improved biomarker performance. The authors of this study agree that ddPCR represents a promising way forward for development of microRNA-based diagnostic assays.
Next-generation sequencing (NGS)
The development of NGS technologies has led to a tremendous amount of accessible and affordable genetic information. NGS promises to revolutionize personal health care as it becomes an integral part of clinical diagnostics. Proper sample preparation prior to sequencing is critical since underloading or overloading at the hybridization step (surface or on beads) can cause low read levels or completely uninterpretable results. Currently, libraries are quantified using qPCR and/or microfluidics-based electrophoresis, but neither method provides sufficient resolution to accurately determine library concentration.
The QX100™ Droplet Digital™ PCR system (Bio-Rad Laboratories, Hercules, CA) is able to maximize sequencing information from NGS platforms as a result of its ability to accurately measure concentration via ddPCR. Its quantitative process permits unbiased and absolute quantification. ddPCR is easy to integrate into the sequencing work flow and can be used for single library quantification as well as for balancing multiple libraries to be run in a single experiment.
As science accelerates, droplet digital PCR will continue to be at the forefront of discovery and will allow researchers to go places they never knew were possible.
- Boettger, L.M.; Handsaker, R.E. et al. Structural haplotypes and recent evolution of the human 17q21.31 region. Nature Genetics July 2012, 1–5.
- Porensky, P.N.; Mitrpant C. et al. A single administration of morpholino antisense oligomer rescues spinal muscular atrophy in mouse. Human Molecular Genetics2012, 21(7), 1625–38.
Frank Bizouarn is an International Field Application Specialist, Bio-Rad Laboratories, Inc., 2000 Alfred Nobel Dr., Hercules, CA 94547, U.S.A.; tel.: 510-741-1000; e-mail: frank_bizouarn@ bio-rad.com.