Journal of Animal Reproduction and Biotechnology 2019; 34(4): 259-266
Published online December 31, 2019
Copyright © The Korean Society of Animal Reproduction and Biotechnology.
Department of Applied Animal Science, Kangwon National University, Chuncheon 24341, Korea
Emulsion polymerase chain reaction (PCR) is performed on compartmentalized DNA, allowing a large number of PCR reactions to be carried out in parallel. Emulsion PCR has unique advantages in DNA amplification. It can be applied in many molecular biological assays, especially those requiring highly sensitive and specific DNA amplification. This review discusses the principle of emulsion PCR and its applications in biotechnology. Related technologies are also discussed.
Keywords: DNA enrichment, droplet digital PCR, emulsion PCR, polymerase chain reaction, SELEX
Polymerase chain reaction (PCR) is an in vitro biological process that mimics in vivo DNA replication, and has led to breakthroughs in biochemistry, biology, and medical science. PCR and associated technologies enable early diagnosis of infectious diseases, rapid prognostic staging for various cancers, sequencing of DNA and RNA, and screening and development of antitumor agents (Viljoen et al., 2005; Hayat, 2008). PCR is expected to continue to play significant roles in advancing bioscience.
The primary aim of PCR and associated technologies is to replicate and amplify target DNA sequences on DNA templates of interest surrounded by nontarget DNA molecules. PCR is carried out via interactions between PCR components, including DNA template, polymerase, primers, nucleotides, and minerals. PCR can identify fewer than ten target DNA sequences among millions of nontarget DNA molecules. However, PCR is hampered by nonspecific reaction between primers and nontarget DNA and primers, resulting in false negatives in infectious disease or cancer testing (Viljoen et al., 2005; Pelt-Verkuil et al., 2008). DNA enrichment, primarily based on PCR, is a necessary step in metagenomic sequencing and systematic evolution of ligands by exponential enrichment (SELEX). Low-frequency DNA variants may be lost during the enrichment of metagenomic specimens and DNA aptamers if a method to replicate DNA sequences independently is not implemented (Fig. 1) (Shanks et al., 2012; Bayat et al., 2018). The occurrence of nonspecific PCR in early stages of the PCR process reduces the amplification of target DNA, which causes false negative results in PCR-based infectious disease and cancer testing, as well as the loss of low-frequency DNA variants.
Schematic illustrates of conventional PCR and emulsion PCR.
PCR in aqueous droplets emulsified in the oil phase of water-in-oil emulsions, termed “emulsion PCR,” has unique attributes for DNA amplification (Kanagal-Shamanna, 2016). Emulsifying the aqueous PCR phase in the oil phase can compartmentalize individual DNA molecules, creating independent PCR environments (Fig. 1). The sensitivity and specificity of PCR can be significantly improved by performing PCR in water-in-oil emulsion (Chai and Oh, 2015; Du et al., 2019). The actual number of pathogens present in clinical specimens can be measured using emulsion PCR-based techniques (Mu et al., 2015). Emulsion PCR-based methods are also used in next-generation sequencing (NGS), metagenomics sequencing, and SELEX to enrich genomic DNA and cDNA libraries while maintaining the same proportions as the original libraries (Shanks et al., 2012; Kanagal-Shamanna, 2016; Bayat et al., 2018). The application of emulsion PCR is expected to support further advances in biological techniques. However, current emulsion PCR-based technologies have several issues that remain to be resolved. For example, emulsion PCR requires the precise optimization of PCR conditions (Kanagal-Shamanna, 2016). Moreover, PCR emulsions can lose their stabilities during temperature fluctuations in PCR.
This review addresses the principle of emulsion PCR and its applications in biotechnology, as well as PCR components and procedures. Technical difficulties associated with emulsion PCR are discussed, and emulsion PCR-based technologies are reviewed.
DNA extracted from a clinical specimen for the diagnosis of infection may include only a few viral or bacterial DNA molecules among millions of host DNA molecules (Strain et al., 2013; Kralik and Ricchi, 2017). The size of genomic DNA and cDNA libraries frequently exceeds 1 × 106 (Ehrlich et al., 2011; Illumina, 2013). The relative abundance of nontarget DNA can cause false negatives in conventional PCR-based diagnostic testing and loss of low-frequency DNA variants in conventional PCR-based DNA enrichment (Fig. 1). Compartmentalizing individual DNA molecules in aqueous PCR droplets emulsified in the oil phase isolates target DNA from nontarget DNA, avoiding some of the challenges associated with conventional PCR-based assays (Fig. 1).
PCR components such as template DNA, polymerase, primers, nucleotides and minerals are soluble in water but not in oil. Water is the continuous phase of the PCR solution, and has different surface energy properties from oil. Thus, oil and water are immiscible. Surfactants can mediate water and oil molecules, allowing water to be dispersed in oil. Tween 80 (polyoxyethylene sorbitan monooleate), Span 80 (sorbitan monooleate), Triton X-100 (polyethylene glycol
Depending on the specific emulsion PCR method, a 20-200 μL volume of the aqueous PCR phase is added to the oil phase to prepare a 100-300 μL PCR emulsion (BioRad.; Chai and Oh, 2015; Witt et al., 2017). Vigorous mechanical agitation of PCR emulsions may generate 108-109 irregularly sized aqueous PCR droplets with a mean diameter of 4-6 μm (Shao et al., 2011; Witt et al., 2017). Droplet diameter and number can be controlled precisely using microfluidic devices (Byrnes et al., 2018). Microfluidic droplet generators are used in emulsion-based digital PCR, referred to as droplet digital PCR (Strain et al., 2013). Typically, microfluidic droplet generators can generate 1 × 105 aqueous PCR droplets with a diameter around 120 μm (BioRad.).
The 100 μL aqueous PCR phase used for emulsion PCR may include target and nontarget DNA molecules, 1.25 U polymerase, 0.8 μM (80 pmol per reaction) of forward and reverse primers, 0.2 mM dNTPs (20 nmol per reaction), and other components (Chai and Oh, 2015; Du et al., 2019). For a 94 kD polymerase, a typical unit of polymerase contains about 8 × 1010 polymerase molecules (Spangler et al., 2009). Thus, 100 μL of the aqueous PCR phase may contain 1 × 1011 polymerase molecules, 4.82 × 1013 forward and reverse primers, and 1.20 × 1016 dNTPs. Assuming that equally sized aqueous PCR droplets are dispersed in the oil phase, the frequency (
Furthermore, the proportion of the sum of individual PCR components compartmentalized by
where λ is calculated by dividing
The proportion (
Emulsion PCR is performed via aqueous droplet generation, PCR amplification inside aqueous droplets, and detection of PCR inside aqueous droplets. Emulsion PCR can be categorized into conventional and fluorescence-based methods. Conventional emulsion PCR includes an emulsion-breaking step to collect enriched DNA molecules, and conventional PCR is subsequently performed with collected DNA molecules from the emulsion. Fluorescence-based emulsion PCR uses fluorescent dyes to directly detect DNA amplification inside droplets.
Conventional emulsion PCR methods generally use mechanical agitation techniques such as stirring and vortexing to emulsify PCR components. PCR emulsions can be prepared using two different formulas. Williams et al. reported an emulsion PCR formulation based on a mixture of 200 μL of aqueous PCR phase (containing PCR components) and 400 μL of mineral oil supplemented with 4.5% Span 80, 0.4% Tween 80, and 0.05% Triton X-100 (Williams et al., 2006). Diehl et al. and Schütze et al. used a formulation composed of 150 μL of aqueous PCR phase and 600 μL of oil phase composed of 73% emollient Tegosoft DEC, 20% mineral oil, and 7% ABIL WE 09 (Diehl et al., 2006; Schütze et al., 2011). In both formulas, bovine serum albumin is included in the aqueous PCR phase to prevent denaturation of the polymerase at the water-oil interface (Diehl et al., 2006; Williams et al., 2006). The
Fluorescence-based emulsion PCR methods include fluorescent dyes, such as SYBR green and EvaGreen, or fluorescence probes such as TaqMan probes, in the aqueous PCR phase to generate fluorescence during PCR inside aqueous droplets (Yang et al., 2014; Martinez-Hernandez et al., 2019; Zhang et al., 2019). Because fluorescence-based emulsion PCR methods are capable of visualizing DNA amplification inside individual aqueous droplets, they have been applied to digital PCR techniques (Strain et al., 2013). Fluorescence-based emulsion PCR methods generally use microfluidic devices to generate aqueous droplets (BioRad.). Microfluidic and fluorescence-based emulsion PCR platforms are now commercially available, such as the QX200 Droplet Digital PCR System (Bio-Rad, Hercules, CA, USA). This commercialized system uses silicone oil and fluorinated oil for the oil phase, as well as surfactants. It is operated with ~1 × 105 aqueous PCR droplets with 120 μm in diameter that are generated with 20 μL of aqueous PCR phase and 70 μL of oil phase by a microfluidic droplet generator (BioRad.). The final concentration of dNTPs recommended by the droplet digital PCR system's manufacturer is 0.8 mM. Under these conditions, the average
Conventional and fluorescence-based emulsion PCR methods may be performed under regular PCR conditions but with a greater number of cycles. The viscosity of the oil phase changes with temperature. As temperature increases and decreases during PCR, aqueous PCR droplets circulate, collide, and are prone to coalesce in PCR emulsions (Holtze et al., 2008). The coalescence of aqueous PCR droplets may compromise the Poisson distribution and emulsion PCR performance. The coalescence of aqueous PCR droplets during PCR can be minimized if PCR is carried out isothermally. The use of loop-mediated isothermal DNA amplification in fluorescence-based emulsion PCR methods confers superior amplification efficiency and specificity compared with previous methods (Ma et al., 2018). Moreover, since isothermal DNA amplification-based methods do not require a thermal cycler, they may support the development of simple, miniaturized fluorescence-based emulsion PCR systems.
Metagenomic NGS is used to investigate the taxonomic composition and genetic content of microorganisms in foods, as well as biological and environmental specimens (Quince et al., 2017; Chiu and Miller, 2019). DNA variants may be present in microbial DNA specimens at frequencies too low for NGS. Thus, DNA variants in some specimens must be enriched before sequencing. DNA enrichment using conventional PCR is biased towards the amplification of short- or high-frequency DNA variants and DNA sequences that match the polymerase sequence preference (Blind and Blank, 2015; Yufa et al., 2015). For example, assuming that a specific DNA variant is 1.5 times as abundant as another DNA, it will become 1.530 = 191,751.1 times as abundant after 30 PCR cycles. DNA enrichment for metagenomics sequencing should amplify DNA molecules in the same proportions as in the original microbial DNA specimen, which can be achieved via emulsion PCR (Kihana et al., 2013). Emulsion PCR is therefore included in many NGS methods for unbiased DNA enrichment (Linderholm, 2019).
Emulsion PCR is also the standard method for enrichment of DNA or cDNA aptamer candidates during SELEX (Witt et al., 2017). SELEX is a molecular biological technique for selecting DNA or cDNA aptamers of interest from an aptamer library comprising ~1 × 1015 sequences (Tuerk and Gold, 1990). Due to the enormous size of the aptamer library, the selection and enrichment of aptamer candidates should be repeated until the aptamer of interest is identified. In conventional PCR, low-frequency DNA or cDNA variants can be lost during enrichment. Enrichment of DNA aptamer candidates using emulsion PCR can prevent the production of PCR byproducts and avoid the loss of low-frequency aptamer candidates (Shao et al., 2011).
Droplet digital PCR has attracted great interest for the early diagnosis of infectious diseases and rapid prognostic staging of cancers. For instance, a droplet digital PCR-based assay capable of quantifying 1 - 104 E. coli in 1 mL of blood has been reported (Kang et al., 2014). Droplet digital PCR-associated diagnostic methods for viruses are more sensitive than other viral diagnosis methods (Strain et al., 2013; Myerski et al., 2019), and may be able to measure a single virus in a clinical specimen (Mu et al., 2015).
The quantity of circulating tumor DNA can be the basis for cancer prognosis and recurrence prediction (Perkins et al., 2017), and is also used to investigate the response and resistance to therapeutic agents (Perkins et al., 2017). However, it is difficult to measure circulating tumor DNA using current molecular biological methods, such as quantitative PCR, because of background DNA from normal cells (Perkins et al., 2017). A recent study showed that a droplet digital PCR-based assay exhibited 13% greater sensitivity than NGS-based assays for KRAS mutations (Demuth et al., 2018). Droplet digital PCR-based circulating tumor DNA assays may be a powerful approach for evaluating cancer patients' prognosis and response to treatment.
No potential conflict of interest relevant to this article was reported.
This study was supported by a grant from the National Research Foundation of Korea (NRF-2013R1A1A2060458), funded by the Korean government.
C Chai, Kangwon Nat'l Univ., Assistant Professor,