Polymerase chain reaction (PCR) was first developed in 1983 by an American biochemist named Kary Mullis. This biochemical method aims to amplify small DNA samples from biological source materials and produce millions to billions of copies of that specific DNA sequence. The process allows for effective sequencing, cloning, and analysis since scientists are not limited to tiny amounts of DNA. Many different variants of PCR exist: assembly PCR, asymmetric PCR, hot start PCR, inverse PCR, and more. Digital polymerase chain reaction (and, more specifically, droplet digital PCR) is the result of biotechnological refinement of conventional PCR methods. It can be used to directly quantify and amplify nucleic acids in DNA, cDNA, and RNA. To truly understand the potential and advantages of digital droplet PCR (ddPCR), one needs to learn the basics of digital PCR (dPCR), how ddPCR works, why it is superior to other methods of PCR, and what its most common applications are.
Digital PCR allows for absolute quantification of target sequences. Conventional PCR is only semi-quantitative because the final, amplified product of this method has to be detected by agarose gel electrophoresis. Real-time PCR (qPCR) is relatively quantitative. It utilizes fluorescence-based detection to measure the amount of the accumulated amplified product throughout the reaction. However, qPCR requires normalization to controls (such as a standard curve or a reference), which does not allow for absolute quantification. Digital PCR has a higher sensitivity than qPCR and enables sensitive detection of rare sequences, such as single-nucleotide mutations in the midst of wild-type DNA sequences. Real-time PCR becomes overwhelmed with wild-type sequences and cannot locate the signal of the rare sequence. Digital PCR overcomes this by minimizing competition between targets in a step called partitioning. Sample partitioning in dPCR represents the process of dividing each sample into a discrete subunit before the PCR amplification begins. Ideally, each partition contains either zero or one template molecule. (Not more than a few.) These partitions behave as individual PCR reactions and fluorescent probe sets are used to identify amplified target DNA (much like in qPCR). Samples that contain the amplified product are considered positive (fluorescent), and those that have no product are negative (with little or no fluorescence). The basis of dPCR quantification lies in the ratio between the positive and negative samples. Instead of following the number of amplification cycles to calculate the initial amount of template sequences in each sample, dPCR relies on Poisson statistics for its absolute template quantity.
Droplet Digital PCR
Compared to PCR that was invented in the 1980s and digital PCR that surfaced in the 1990s, ddPCR only became commercially available in 2011. It is one of the most recent technologies for cloning, amplifying, and quantifying nucleic acid sequences. Digital droplet PCR is a subtype of digital PCR that utilizes a water-oil emulsion droplet system. These emulsion droplets represent partitions and serve to separate template DNA molecules. They essentially have the same function as individual test tubes although on a much smaller scale. The key aspect of ddPCR is this massive sample partitioning. So, how does ddPCR work? The sample is partitioned into twenty thousand nanoliter-sized droplets (the ideal droplet number). The division is achieved through a combination of surfactant chemistries and microfluidics. The PCR amplification process develops through a series of workflows and with reagents similar to those used in standard TaqMan probe-based assays. Once the PCR is complete, each nanoliter droplet is analyzed to determine whether it contains the product or not (whether they are positive droplets or negative droplets). The collected data is then run through Poisson statistics to determine the concentration of the target template in the original sample.
ddPCR vs. qPCR
When considering the advantages of ddPCR over other PCR variants, ddPCR is most commonly compared to qPCR because they are similar methods. Both variants use fluorescence and are quantitative measures. However, qPCR measures the fluorescence of droplets at specific moments in the process (usually after every amplification cycle, which requires stops in the experiment). It determines the relative amount of the nucleic acid targets but cannot specify the precise amount without using a standard curve. Real-time PCR provides a value called CT (threshold per cycle). The difference between CTs of different cycles is used to find the amount of initial target sequence. Because of this, qPCR is considered an ‘analog’ measurement and not nearly as precise as ddPCR. While qPCR measures the quantity of the nucleic acid during the amplification process, ddPCR only does so after it is complete. It is an endpoint measurement since the data is observed only after the experiment is finished. ddPCR is less susceptible to impaired amplification efficiencies. Furthermore, real-time PCR cannot distinguish between copy number variations or gene expressions smaller than 2-fold. Compared to that, ddPCR has a much higher precision rate. Studies have shown that digital droplet PCR distinguishes between copy number variations that vary by only 1 copy, detects differences when they are less than 30% in gene expression, and allele detection for alleles that occur less frequently than 0.1%.
ddPCR vs. Other dPCR Technologies
Digital PCR technology has its advantages, but it also has its shortcomings. dPCR techniques aren’t scalable and lack practicality. Samples require serial dilution, which is a laborious process with a probable chance of error in pipetting. Chip-based systems are also time-consuming, requiring complex fluidic schemes for the partitioning step. ddPCR overcomes these issues by making the partitioning process incredibly simple. Only one step is required for massive partitioning of one sample in the fluid phase. Tens of thousands of droplets in ddPCR mean that one sample can create tens of thousands of data points instead of a single result. With the right ddPCR system, this droplet generation, thermal cycling, droplet reading, and data analysis can be accessible to any working research laboratory.
Benefits of ddPCR
- Absolute quantification. ddPCR is perfect for measuring target DNA, quantifying microbes, and analyzing viral loads. It provides an absolute count of the copies of target sequences per sample without the need for including standard curves.
- Simple quantification process. Because you don’t need any reference (ΔΔCq method) or calibration standard, ddPCR makes the absolute quantification process incredibly straightforward and simple.
- Outstanding precision. Thanks to massive sample partitioning, this PCR technique allows for precise measurement of small fold differences in target nucleic acid sequence copies between samples. This is especially beneficial in genetic material testing.
- Improved signal-to-noise ratio. ddPCR is highly sensitive, allowing for the detection of rare targets. Its quantification has a precision of ±10%. This is possible due to the high target DNA concentration in positive droplets because the background and high-copy templates are diluted.
- No PCR bias. Unlike qPCR, there is no amplification efficiency reliance in ddPCR. It can detect small differences (1.2-fold), thus reducing error rates.
- Reduced costs. For this analysis, the quantity of reagents and samples is reduced to a minimum. The reaction volumes are in the range of pico- and nanoliters, effectively lowering the costs of the process. Furthermore, ddPRC is emulsion-based, meaning that its equipment doesn’t require any microfluidics or complex chips. It can be performed in a standard thermal cycler.
- Superior partitioning and accuracy. Digital PCR techniques based on chips produce only hundreds or thousands of partitions. ddPCR is superior in this sense, yielding twenty thousand droplets in a sample volume of 20 µl. This higher number of partitions leads to higher accuracy of the end result.
Overall, ddPCR is more efficient, more accurate, and easier to perform than other forms of PCR, including other dPCR technology.
Applications of ddPCR
Ever since the first ddPCR machines were developed, scientists have been trying to introduce the process into a clinical setting. qPCR cannot be a diagnostic standard because it has low positive and sometimes even false positive outcomes in clinical samples. However, ddPCR is more accurate and more sensitive than qPCR, allowing for more opportunities. So, which applications or assays can be performed using ddPCR? This PCR type can be applied in the following areas:
- Measuring levels of gene expression – Even though qPCR is an excellent method for detecting 2-fold or greater gene expression differences above 100 copies, there are some challenges in this process. Namely, qPCR cannot minimize the influence of contaminants while still maintaining quantifiable levels of the nucleic acid. This is where ddPCR is superior – it allows quantification of these expression differences (and smaller!) with greater sensitivity and precision.
- Determining copy number variation – Measuring the copy number variation is essential in multiple disciplines. Genomic segments between the members of the same species can vary in copy number, and between normal and cancer cells of the same individual. ddPCR’s partitioning method enables accurate measurement of copy number variation in genetic studies.
- Detecting rare DNA target copies – Rare mutation detection (RMD) represents the detection of a DNA sequence that is present at a very low frequency in a wild-type background. The issue for most PCR techniques is that wild-type and target sequences are incredibly similar, with the wild-type droplets being significantly more abundant. However, ddPCR easily overcomes this issue. RMD assays are typically used in examining samples of cancer biopsies.
- Next-generation sequencing (NGS) – ddPCR can measure NGS sample library preparations to increase the accuracy of the sequencing and reduce run repeats. It can be used to validate sequencing results such as polymorphisms of a single nucleotide.
- Single-cell analysis – Genomic content in progenitor, homogeneous post-mitotic, and stem cell populations can be varied to a high degree (10- to 100-fold). ddPCR allows quantification of low copy numbers and analyzing these cell-cell variations.
- Genome edit detection – If a genome-editing tool is used, ddPCR offers a cost-effective, fast, and precise assessment of its results.
Infectious Disease Diagnosis
ddPCR is a powerful tool in detecting the pathogens causing infectious diseases (viruses, bacteria, and parasites). Here are some examples of diseases and pathogens that can be seen by ddPCR:
- Malaria (malaria parasites, Plasmodium)
- Tuberculosis (Mycobacterium tuberculosis)
- Staphylococcus, Salmonella, and Listeria bacterial infections
- Hepatitis B (hepatitis B virus, HBV)
- Horse herpes (equine herpesvirus 1 and 4)
- Acquired immunodeficiency syndrome, AIDS (human immunodeficiency virus, HIV)
Digital droplet PCR enables easier sampling in some diagnoses that were hard to determine in the past, such as extrapulmonary tuberculosis. It is more suitable for clinical usage than qPCR, and it also provides faster results (readouts) than pathogen culturing or serological tests.
ddPCR is effectively utilized in oncology in two ways: for analyzing archival tumor tissues and liquid biopsies.
- Archival tumor tissues – These types of tissues are the most easily accessible source for biomarker testing. However, the DNA quality in them can be poor, and the sample availability is limited. ddPCR has proven helpful in such cases, where not much of the sample is required to achieve the necessary quantities for analysis.
- Liquid biopsies (blood samples, cerebrospinal fluid, BM, tears, urine, saliva, cfRNA, cfDNA) – Compared to standard tumor biopsy, liquid biopsy is less invasive and more convenient for both patients and medical practitioners. It can more accurately describe the heterogeneity inside a tumor and track the changes that occur in response to treatment during follow-ups. ddPCR is successfully used in diagnosing, predicting, and monitoring a large number of malignancies.
As previously established, ddPCR is unparalleled when it comes to analyzing low abundance or challenging samples. If you need analysis of cell-free DNA or environmental DNA and minute gene expression fold changes or locating rare genetic events in mixed cell populations – ddPCR is your process. When choosing your ddPCR service provider, this is what you need to consider:
- Turnaround times – You should not have to wait too long to receive the results you’re after. The provider you choose should be in regular contact and transparent with the project deliverables and deadlines.
- Quality results – If the reports you receive are tainted in any way, or have doubts about the quality or integrity of the process, you will have no choice but to repeat the analysis. Your ddPCR service provider should have a robust quality management system and deliver complete documentation on the laboratory procedures.
- Industry-leading technology – The current gold standard for ddPCR systems is Bio-Rad QX200 Droplet Digital PCR System, which, incidentally, MOgene happens to use as well.
- CLIA-certified facility – CLIA stands for Clinical Laboratory Improvement Amendments. Every laboratory that accepts human samples for testing (regardless of the sample type) needs to be CLIA-certified and following all the proper national and international compliance requirements.
- Data security – Your ddPCR provider should make confidentiality a top priority. Consider whether they have secured data records, how often they back up their information, if they monitor their humidity and temperature conditions, and whether their labs are key-coded.
- BioSafety practices – If you’re working in R&D for vaccines or any other type of clinical study involving pathogens, you have to go for a provider that has BSL-2+ facilities.
- Project aspects – How much of the project will your company have to handle, and how much will the ddPCR provider take on? An analysis project usually includes assay design, nucleic acid isolation, assay optimization, data analysis, and detailed reporting. Some service providers can handle every aspect of the project, so factor that into your selection process.
There is no doubt that PCR is an invaluable tool in modern-day biotechnology. Without it, scientists would never be able to analyze and work with limited amounts of DNA samples. Through PCR history, this method has evolved from a semi-quantitative to relatively quantitative analysis with real-time PCR (qPCR). Finally, it reached the point of absolute quantification with digital PCR (dPCR). Droplet digital PCR (ddPCR) is a form of dPCR that delivers precise and accurate results with incredible sensitivity. It is scalable and practical, as its partitioning step does not require too much time or effort. The entire concept of ddPCR is that the sample is divided into 20,000 nanodroplets. After PCR amplification is complete, these droplets are assessed in a droplet reader for the presence or absence of the target DNA molecule. Based on the number of positive droplets, one can easily calculate the amount of the target sequence in the original sample. ddPCR is more cost-effective than other PCR technologies. It can be used in various ways, including determining gene expression differences, copy number variations, single-cell analysis, NGS, and more. It has taken its rightful place in oncology for analyzing liquid biopsy samples, and immunology for detecting pathogens of infectious diseases. If you are interested in reaching out to a ddPCR service provider for your trials or testing, make sure they meet all the required standards. Don’t hesitate to contact our experts at MOgene for more information on ddPCR, how it can help your project, and what we can do to make the process as easy for you as possible.