Digital polymerase chain reaction

Digital polymerase chain reaction (digital PCR, DigitalPCR, dPCR, or dePCR) is a biotechnological refinement of conventional polymerase chain reaction methods that can be used to directly quantify and clonally amplify nucleic acids strands including DNA, cDNA or RNA. The key difference between dPCR and traditional PCR lies in the method of measuring nucleic acids amounts, with the former being a more precise method than PCR, though also more prone to error in the hands of inexperienced users.^[1]:217 A “digital” measurement quantitatively and discretely measures a certain variable, whereas an “analog” measurement extrapolates certain measurements based on measured patterns. PCR carries out one reaction per single sample. dPCR also carries out a single reaction within a sample, however the sample is separated into a large number of partitions and the reaction is carried out in each partition individually. This separation allows a more reliable collection and sensitive measurement of nucleic acid amounts. The method has been demonstrated as useful for studying variations in gene sequences — such as copy number variants and point mutations — and it is routinely used for clonal amplification of samples for "next-generation sequencing."

PCR basics

The polymerase chain reaction method is used to quantify nucleic acids by amplifying a nucleic acid molecule with the enzyme DNA polymerase. Conventional PCR is based on the theory that amplification is exponential. Therefore, nucleic acids may be quantified by comparing the number of amplification cycles and amount of PCR end-product to those of a reference sample. However, many factors complicate this calculation, creating uncertainties and inaccuracies. These factors include the following: initial amplification cycles may not be exponential; PCR amplification eventually plateaus after an uncertain number of cycles; and low initial concentrations of target nucleic acid molecules may not amplify to detectable levels. However, the most significant limitation of PCR is that PCR amplification efficiency in a sample of interest may be different from that of reference samples. Since PCR is an exponential process, only twofold differences in amplification can be observed, greatly impacting the validity and precision of the results.

dPCR working principle

Figure 1. Oil droplets containing fluorescent PCR target molecule

Figure 2. Fraction of positive droplets predict number of target copies per droplet modeled by the Poisson distribution

dPCR improves upon the current PCR practices by dividing up the reaction into multiple, smaller reactions. A sample is partitioned so that individual nucleic acid molecules within the sample are localized and concentrated within many separate regions. Micro well plates, capillaries, oil emulsion, and arrays of miniaturized chambers with nucleic acid binding surfaces can be used to partition the samples. A PCR solution is made using a Taqman assay, which consists of template DNA (or RNA), Fluorescence-Quencher probes, primers, and PCR mastermix. The PCR solution is divided into smaller reactions and are then made to run PCR individually. After multiple PCR amplification cycles, the samples are checked for fluorescence with a binary of “0” and “1”. The fraction of fluorescing droplets is recorded. The partitioning of the sample allows one to estimate the number of different molecules by assuming that the molecule population follows the Poisson distribution, thus accounting for the possibility of multiple target molecules inhabiting a single molecule. Using Poisson's Law of small numbers, the distribution of target molecule within the sample can be accurately approximated allowing for a quantification of the target strand in the PCR product.^[2] Figure 2 shows the Poisson distribution of the copies of target molecule per droplet (CPD) based on the fraction of fluorescent droplets (p), represented by the function CPD=-ln(1-p).^[3] This model simply predicts that as the number of samples containing at least one target molecule increases, the probability of the samples containing more than one target molecule increases. In conventional PCR, the number of PCR amplification cycles is proportional to the starting copy number. dPCR, however, is not dependent on the number of amplification cycles to determine the initial sample amount, eliminating the reliance on uncertain exponential data to quantify target nucleic acids and therefore provides absolute quantification.

The benefits of dPCR include increased precision through massive sample partitioning, which ensures reliable measurements in the desired DNA sequence due to reproducibility. With basic PCR, error rates are larger when detecting small fold differences, however, with dPCR the error rate decreases because smaller fold differences can be detected in DNA sequence. The technique itself reduces the use of a larger volume of reagent needed, which inevitably will lower experiment cost. Also, dPCR is highly quantitative as it does not rely on relative fluorescence of the solution to determine the amount of amplified target DNA.

Digital-Droplet PCR

Figure 3. Representation of oil emulsion technique used in digital droplet PCR

In Digital Droplet PCR (ddPCR) the PCR solution is divided into smaller reactions through a water oil emulsion technique, which are then made to run PCR individually. As shown in Figure 3, the PCR sample is partitioned into nanoliter-size samples and encapsulated into oil droplets.^[4] The oil droplets are made using a droplet generator that applies a vacuum to each of the wells.^[5] Approximately 20,000 oil droplets are made from each 20 μL sample.

Comparison of dPCR and Real-time PCR (qPCR)

dPCR measures the actual number of molecules (target DNA) as each molecule is in one droplet, thus making it a discrete “digital” measurement. It provides absolute quantification because dPCR measures the positive fraction of samples, which is the number of droplets that are fluorescing due to proper amplification. This positive fraction accurately and precisely indicates the initial amount of nucleic acid (template DNA). Similarly, qPCR utilizes fluorescence; however, it measures the intensity of fluorescence at specific times to determine the relative amount of target molecule (DNA), but cannot specify the exact amount. It gives the threshold per cycle (CT) and the difference in CT is used to calculate the amount of initial nucleic acid. As such, qPCR is an analog measurement, which is not as precise due to the extrapolation required to attain a measurement.^[6]

Also, dPCR measures the amount of DNA after amplification is complete and then determines the fraction of replicates. This is representative of an endpoint measurement as it requires the observation of the data after the experiment is completed. In contrast, qPCR records the relative fluorescence of the DNA at specific points during the amplification process, which requires stops in the experimental process. This “real-time” aspect of qPCR can affect results occasionally due to the stopping of the experiment.

More specifically, qPCR is unable to distinguish differences in gene expression or copy number variants that are smaller than twofold. It is difficult to identify alleles with frequencies of less than 1% because highly abundant, common alleles would be matched with similar sequences. On the other hand, dPCR has been shown to detect a differences of less than 30% in gene expression, distinguish between copy number variations of that differ by only 1 copy, and identify alleles that occur at frequencies less than 0.1%.^[7]

Application

dPCR application can include, but is not limited to, detection of genetic-based disease. For example, it can be used to identify a rare allele in a developed heterogenous tumor. In addition, the technique can be used for genetic prenatal diagnostics. In a research paper conducted by White et al., a dPCR assay was able to detect an L1 insertion event in as few as 0.01% to 0.1% of cells, which is an example of how specific and accurate dPCR can be.^[8]

dPCR provides quantification of gene expression levels, especially with low-abundance miRNA due to the technique's sensitivity and precision. dPCR can be used for amplification of all types of RNA such as siRNA, mRNA, etc. Due to the high degree of cell-cell variation in gene expression, dPCR enable low copy number quantification in order to perform single cell analysis. Following variations among cells, dPCR enables an increased detection of differences in gene copy number in order to analyze complex behavioral traits, phenotypic variability, and diseases.^[9]

Development

The first dPCR paper was published by Dr Alec Morley and Pamela Sykes in 1992. The purpose was to quantify PCR targets in an attempt to track and measure the absolute lowest number of leukemic cells in a patient with leukemia. The purpose was to monitor residual disease in leukemia patients, and thereby treat the patients at the earliest possible moment of detection of disease recurrence. Further evolutions of the technology allowed for more widespread distribution of this technique, with small partitions created by emulsion droplets and/or microfluidics.^[10]

In 1995, Brown at Cytonix and Silver National Institutes of Health coinvented single-step quantitization and sequencing methods employing nano-scale physical containment arrays (Brown, Silver),^[11] and open chambers (Brown) using localized clonal colonies in 1D and 2D capillaries, macro volumes, gels, free chambers, and affinity surfaces/particles. resulting in a 1997 U. S. Patent,^[12] and subsequent divisional and continuation patents. The concepts of electrowetting and digital microfluidics were further introduced (Brown) as one means of manipulating nano fluid volumes.

Digital PCR has been shown to be a possible surveillance tool for illnesses such as cancer, and as a vital front end to determining genomic content, including sequencing the human genome.

Vogelstein and Kinzler developed a technology called BEAMing based on digitial PCR (Beads, Emulsion, Amplification, Magnetics) and quantified KRAS mutations in stool DNA from colorectal cancer patients.^[13][14]

Dressman, et al, began using emulsion beads for digital PCR,^[15] and dPCR has also proved useful for the analysis of heterogeneous methylation.^[16]

In 2006 Fluidigm introduced the first commercial system for digital PCR based on integrated fluidic circuits (chips) having integrated chambers and valves for partitioning samples.

In 2008, Inostics started to provide BEAMing digital PCR services for the detection of mutations in plasma/serum and tissue.

QuantaLife developed a different method of partitioning, called the Droplet Digital PCR (ddPCR) technology, which partitions a sample into 20,000 droplets and digitally counts nucleic acid targets. In 2011, Bio-Rad Laboratories acquired Quantalife.^[17]

In 2013, RainDance Technologies launched a digital PCR platform based on its picoliter-scale droplet technology, which generates up to 10 million picoliter-sized droplets per lane.^[18][19] The technology was first demonstrated in a paper published in Lab on a Chip by scientists from Université de Strasbourg and Université Paris Descartes.^[19][20] Later that year, RainDance Technologies announced a partnership with Integrated DNA Technologies to develop reagents for the digital PCR platform.^[21]

Digital PCR has many potential applications, including the detection and quantification of low-level pathogens, rare genetic sequences, copy number variations, and relative gene expression in single cells. This method provides the information with accuracy and precision. Clonal amplification enabled by single-step digital PCR is a key factor in reducing the time and cost of many of the "next-generation sequencing" methods and hence enabling personal genomics.

MIQE

The "Minimum Information for Publication of Quantitative Digital PCR Experiments" or "digital MIQE" guidelines are a comprehensive set of best practices which aim to increase the validity and comparability of digital PCR experiments reported in published literature.^[1]:217 The guide was published in 2013 and followed publication in 2009 of "MIQE", a comparable guide for quantitative real-time PCR.^[22][23] In the two years following publication of the "digital MIQE", less than 20% of published digital PCR papers have cited the guideline.^[1]:217

References

External links

• Digital PCR Protocol

Reviews