History of Digital PCR Trisha Dhawan and Rémi Dangla 8/8/19

Outline 1.1. PCR, the start of a revolution ...... 1 1.2. Limit dilution PCR ...... 1 1.3. Digital PCR, a term coined by Vogestein and Kinzler ...... 2 2.1. PCR meets microfluidics ...... 2 2.2. PCR in micro-droplets ...... 3 3.1. Digital PCR in Microarrays ...... 4 3.2. Digital PCR in Micro-droplets ...... 4 Combining microarrays and microdropelts with Crystal™ digital PCR ...... 5 Discussion ...... 6

The origins of Digital PCR of template nucleic acid molecules, oligonucleotide primers, dNTPs, and a 1.1. PCR, the start of a revolution thermostable DNA polymerase. Over the years, The polymerase chain reaction (PCR) was ongoing development and application of the PCR invented by Kary Mulis while he was developing reaction enabled molecular cloning, engineered methods for detection of point mutations using transgenic organisms, DNA forensics, clinical oligonucleotides at Cetus Corporation, diagnostic DNA sequencing, among many California. The idea of PCR occurred to him additional innovative technologies. while driving through the Californian mountains In a traditional PCR reaction, the template on a Friday night in 1983. Contemplating the idea DNA is usually several nanograms’ worth that occurred to him, he reasoned that using two corresponding to millions of DNA molecules. oligonucleotides of different lengths instead of The template DNA is most often a heterogeneous one to bind to the upper and lower strands of the mixture of mutated alleles, plasmids, or genomes target DNA sequence and copying it several times depending on the application. Cancer is one of the using a polymerase enzyme would amplify the clinically relevant examples of this heterogeneity, target sequence manifold, and could then be where a relatively small quantity of template separated on a gel. He developed the method over DNA originating from cancer cells may harbor a the next few months using different temperatures, cancer-causing somatic mutation relative to the polymerases, and most importantly cycling the wild-type (WT) DNA. reaction multiple times, 30 cycles, to strengthen the signal without a background1. He later 1.2. Limit dilution PCR patented the technology and in 1987, Thermal In the early 1990s, several research groups began Cyclers were made commercially available by a exploring the possibility of diluting the template joint venture between Cetus and Perkin-Elmer. DNA to an extent such that, on average, any PCR forever revolutionized the field of molecular single PCR reaction contained only a single biology and clinical genetics. template molecule, a method named “limit A PCR reaction involves the exponential dilution PCR”. A key advantage of limit dilution amplification of a target polynucleotide sequence PCR is that each DNA molecule is amplified by repeatedly thermocycling a salt-buffered mix separately, killing interferences between template 1

molecules during PCR and greatly reducing PCR allowed the detection and quantification of background noise in complex samples. the targeted mutant DNA sequences in the Additionally, when many reactions are performed partitioned sample at this level of dilution, the frequency of positive Vogelstein and Kinzler named their and negative reactions follows the Poisson method “digital PCR”, in reference to the distribution and therefore allows for calculating classification of reactions as “zeros/negatives” or the abundance of the target molecule based on the “ones/positives” in a similar fashion to bits dilution factor. classification in computer science. 2 In 1992, Sykes et al. first described the concept The two scientists recognized the variety of digital PCR using the principles of limiting of potential applications of digital PCR, including dilution, PCR, and Poisson statistics to quantitate detecting somatic single nucleotide variants in the total number of rare leukemic cells in a cancer, chromosomal translocations, changes in population of normal cells. Other researchers gene expression, allelic discrimination, and allelic reported using versions of limit dilution PCR imbalance. In 1999, they also noted that the limit strategy to study, for example, variation among 3 4 of detection was defined by the number of single- HIV proviruses , human genomic haplotyping , template reactions that could be analyzed, paving and to quantify the fraction of leukemic cells in 5 the way for increased sensitivity of the patient samples . These publications are the first technology by using high-throughput platforms reports of digital PCR, from pioneers who such as 1,536-well plates, microarrays, and developed the method before it was even called beyond. digital PCR. The scientific community quickly recognized key

1.3. Digital PCR, a term coined by advantages of digital PCR over traditional end- Vogestein and Kinzler point or real-time PCR: does not rely on a In 1999, Bert Vogelstein and Kenneth standard curve; has improved accuracy; provides Kinzler6 recognized the urgent importance of absolute quantification; and offers improved detecting cancer-causing somatic mutations at an detection of low copy-number variants. Other key initial stage in clinical samples to enable early advantages of digital PCR became evident later, cancer diagnosis. Indeed, reliably detecting these as the method was used more broadly7: rare somatic mutations could help diagnose repeatability of assays, over time and across primary tumors in patients who are asymptomatic different labs; robustness and tolerance to PCR and whose disease is still curable. They also inhibitors. observed that detecting these mutations depended on isolating them from a large excess of WT DNA. PCR in Microfluidic Systems Vogelstein and Kinzler developed a strategy 2.1. PCR meets microfluidics based on limit dilution PCR to selectively amplify these rare mutations, distinguish them from WT, In the mid-1990s, advances in microfabrication and quantify the fraction of mutant alleles relative techniques had allowed the production of devices to WT. The dilution PCR strategy allowed one to with feature sizes as small a few microns. partition individual template molecules such that Micromachining, photolithography, and etching the resulting amplification was either completely silicon and glass substrates produced networks of mutant or completely WT. In practice, they flow channels that could sort reagents and diluted and partitioned the starting DNA template molecules, partition chemical reactions, and act into a 384-well plate, so that any given well as droplet generators for mixtures of aqueous and contained one-half genome equivalent on oil-based fluids. These microfluidic devices were average, i.e. half the wells contained one template well-suited for performing biochemical assays, molecule and half the wells contained zero such as single-cell assays, studies of template molecules. Subsequent amplification by 2

Figure 1. Reverse micelles in square channels. Photomicrographs show the transition from the 30 mm wide channel to the 60 mm wide channel. Respective pressures for the water and oil/surfactant (hexadecane_2% Span 80) are noted in the figure13. macromolecules e.g. proteins and nucleic acids, annealing, and primer extension temperatures and the polymerase chain reaction (PCR). appropriate for PCR11,12. The partitioning capabilities of In late 1999, the first commercial lab-on- microfluidic lab-on-chip devices were a-chip, the HP 2100 Bioanalyzer, was introduced particularly useful for PCR, where a by Hewlett-Packard’s Chemical Analysis Group, microenvironment allows for increased which shortly thereafter became Agilent. Based thermocycling speeds and increased reagent on Caliper Technologies’ LabChip, the 2100 was concentrations within the reaction mixture8. the first complete instrument that could perform Several groups developed integrated systems sample handling, separation, fragment sizing, utilizing microfabricated components of glass and quantitation, and digital data processing for PCR silicon to perform restriction enzyme digests, products, restriction enzyme digests, or RNA PCR, and electrophoresis within a single chip- samples. Following the HP 2100, other lab-on- based device9,10. These systems generally relied chip platforms were commercialized by on thermocapillary pumps to perform PCR in companies including Nanogen Inc., HandyLab nanoliter-sized droplets within micron-sized Inc., and Micronit Microfluidics among others. channels and included entry ports, heating elements, and DNA detectors. They did not 2.2. PCR in micro-droplets require any novel hardware, biochemistries or By the early 2000s, water-in-oil DNA detection methods; rather, they simply emulsions were being studied for potential miniaturized existing components such as heaters applications designed to partition biological and applied well-known PCR methods in a materials into picoliter-scale droplets13,14. A micron-sized environment on a chip. significant advance was the use of surfactants in By the late 1990s, integrated chips the oil-based continuous phase to maintain the utilizing the continuous flow of nanoliter-sized integrity of droplets formed after the introduction plugs through microchannels were becoming of the discontinuous aqueous phase. Because of common. These systems achieved thermocycling surfactants in the oil-filled microchannels, by transporting the reaction plugs through a droplets can be pooled in widened channels or reservoirs, touching each other without breaking microcapillary etched into a glass chip that 13 traverses heaters at the respective melting, or fusing (Fig.1). Extensive fluidic networks of oil-and-surfactant filled channels can be designed

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to coordinate the intentional mixing of plugs or microfluidic digital PCR in chambers had been droplets to mediate biochemical reactions14. used for applications as varied as interrogating 19 Fluorinated oils combined with microbial community diversity , human genomic copy number variation20,21, and fetal fluorinated surfactants were particularly useful 22 for handling biomolecules within aqueous aneuploidy . droplets, as these carrier fluids are inert, stable at With microfluidic-based digital PCR high temperatures, compatible with the most becoming well-known and more widely common lab-on-chip materials, and can prevent practiced, in 2006 Fluidigm Corporation became surface adsorption of charged biomolecules14–16. the first company to commercialize the Fluorinated oils and surfactants were commonly technology in an integrated microfluidic circuit. used and commercially available, e.g. The BioMark system was based on the 12.765 FluorinertTM by 3M and ZonylTM by Dupont, Digital Array, a chip of 12 panels, each panel among other offerings from Fluorochem Ltd., partitioned into 765 6-nL chambers. After loading Sigma-Aldrich Inc., and others. the PCR reaction mixture through 12 carrier inputs, the chip was thermocycled, fluorescence The combination of commercially available lab-on-chip microfluidic networks with was detected, and the signal was processed and advanced picoliter-scaled aqueous droplet analyzed by the Digital PCR Analysis software. generation, stable surfactant-mediated pooling However, digital PCR technology during this and sorting of droplets provided future time was limited by the number of individual opportunities for single-molecule reactions partitions (chambers) per sample, volume of the within these droplet microreactors. reactions, and increased hands-on time. Moreover, digital PCR was very costly to run, with a reaction costing several hundred dollars Next-Generation Digital PCR compared to a little over a dollar for traditional end-point or real-time PCR. 3.1. Digital PCR in Microarrays 3.2. Digital PCR in Micro-droplets In the early 2000’s, the digital PCR method described by Vogeslstein and Kinzler was largely An alternative approach that further overshadowed by real-time PCR. Indeed, such enhanced throughput and sensitivity while early digital PCR experiments, performed using addressing the cost per reaction limitation was to microtubes or 384-well plates, suffered from generate picoliter-sized microdroplet reactors by drawbacks such as the amount of reagents flow-focusing offering many more partitions than needed, limited capacity for partitioning and the chamber-based systems. Droplets are automation. However, in the engineering and generated in a microfluidic system, thermocycled microfluidics world, digital PCR publications to perform single-molecule digital PCR within were increasing exponentially. Efforts to enhance the droplets, and end-point amplification is sensitivity by partitioning the template molecules detected and quantified via real-time fluorescence into thousands of chambers at nanoliter-scale curves23. These droplet-based lab-on-chip volumes, along with providing the additional systems were also adapted to perform reverse advantages of reagent savings, decreased transcription PCR (RT-PCR) to detect single diffusion distances, and improved statistics were copies of RNA genomes24 and to perform ongoing. A further innovation was multiplexing, multiplex reactions directed to multiple targets i.e. targeting multiple alleles within a single PCR within a single droplet25. mixture by introducing a plurality of primers and 17,18 This technology was first commercialized fluorescent probes . Since target molecules are by QuantaLife, Inc. as the QX100 ddPCR™ partitioned individually, a given primer pair will System in 2011. The microfluidic consumables only amplify its particular target in a partition that used on the ddPCR™ platform could contains that unique allele. By the mid-2000’s,

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Figure 2. Visualization of droplet crystals. Droplet Crystal containing 29078 analyzable droplets, imaged post PCR using the Blue (A), Green (B) and Red (C) Prism3 acquisition channels. The left insert in A. presents a zoomed-in portion of the droplet crystal. Droplets in which no amplification has taken place remain dark, and droplets in which amplification is observed are lighter in color. Droplets have a higher fluorescent baseline in the Blue channel due to the FITC added to the reaction mix26. accommodate up to eight samples per chip, 30,000 droplets that make up the droplet crystal, generating 14,000-16,000 droplets per sample. and a fluorescence read-out is performed at end The reduction in the cost of running point by taking high-resolution images of the crystal. This digital PCR workflow, named digital PCR experiments on the QX100 ddPCR™ System accelerated the adoption of digital PCR Crystal™ digital PCR combining the advantages for many applications in molecular biology. of a) array-based digital PCR such as an Nonetheless, when compared to the standard real- integrated workflow and multiplexing; with b) time or quantitative PCR, the digital PCR those of droplet-based PCR such as reduced cost. workflow remained cumbersome to run, with The ability to partition the sample extensively and high hands-on time, multiple instruments uniformly affords the system with superior required and long time-to-result. Multiplexing precision and sensitity for target detection and capabilities were also limited to two detection quantification. Additionally, the flexibility to channels. explore different fluorophores, the capability of performing multiplexing of targets (up to 3- Combining microarrays and colors26; Figure 2), and a fast time to result make microdropelts with Crystal™ digital it a unique digital PCR system. A recent PCR advancement,as a proof of concept, the capacity to detect multiple targets in Lung cancer patient An advanced digital PCR equipment, the samples using 6-color Crystal™ digital PCR was TM Naica System , was launched in 2016 by Stilla reported, opening new avenues for multiple target Technologies. This system performs digital PCR DNA investingation27. by partitioning the sample, using a confinement gradient (Figure 3), into a large 2D array of droplets, also called a droplet crystal. A PCR reaction occurs in each of the partitioned 25- 5

microfluidics, molecular biology, and computer science fields resulted in significant improvements in the digital PCR machines over the past decade. For many applications, digital PCR is beginning to replace the conventional qPCR owing to its ease-of-use, reduced complexity, repeatability, and superior precision. Now, efforts to further enhance the applicability of the digital PCR technique to a wide range of experiments in different research areas are in place, with the ultimate aim of making digital PCR a standard lab technique. References: 1. Nobel Lecture. NobelPrize.org. Nobel Figure 3. Device geometry and mechanism for Media AB 2019. Wed. 7 Aug 2019. drop formation through a confinement gradient. vailable at: (A) Three-dimensional sketch of a device during https://www.nobelprize.org/prizes/chemis operation. The dispersed phase is pushed through try/1993/mullis/biographical/. (Accessed: the inlet channel (width wand height h0) into a 7th August 2019) wide reservoir containing a stationary continuous phase. The top wall of the reservoir is inclined at 2. Sykes, P. J. et al. Quantitation of targets for an angle α. Fluid from the continuous phase PCR by use of limiting dilution. remains in the corners of the inlet channel, Biotechniques 13, 444–9 (1992). forming gutters connected to the reservoir. (B) 3. Simmonds, P. et al. Human For a flat reservoir (α=0°), the circular tongue grows indefinitely without detaching. (C–E) Even immunodeficiency virus-infected a small slope (α=1.2°) leads to a modification of individuals contain provirus in small the tongue shape and to a drop detaching. (C) A numbers of peripheral mononuclear cells tongue of water in oil has a projected surface area and at low copy numbers. J. Virol. 64, A. (D) A neck appears in the inlet channel and its 864–72 (1990). width w decreases in time. (E) The thread m 4. Ruano, G., Kidd, K. K. & Stephens, J. C. ruptures, when wm=h0, releasing a self-propelled droplet. (F ) Cross-sectional shape of the confined Haplotype of multiple polymorphisms thread in the inlet channel for different imposed resolved by enzymatic amplification of C. If C>C*, the interface flattens against all four single DNA molecules. Proc. Natl. Acad. walls and the gutter radius of curvature r = 1/C. Sci. U. S. A. 87, 6296–300 (1990). For C = C*, r = h0/2: The inner fluid is tangent to 5. Brisco, M. J. et al. Outcome prediction in the side walls. When C

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