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Principle of Emulsion PCR and Its Applications in Biotechnology

Authors:

Abstract

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.
INTRODUCTION
Polymerase chain reaction (PCR) is an
in vitro
biologi-
cal process that mimics
in vivo
DNA replication, and has
led to breakthroughs in biochemistry, biology, and medi-
cal 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 technolo-
gies 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 dis-
ease 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 systemat-
ic evolution of ligands by exponential enrichment (SELEX).
Low-frequency DNA variants may be lost during the en-
richment 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.
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 signifi-
cantly improved by performing PCR in water-in-oil emul-
Principle of Emulsion PCR and Its Applications in
Biotechnology
Changhoon Chai*
Department of Applied Animal Science, Kangwon National University, Chuncheon 24341, Korea
Review Article
J Anim Reprod Biotechnol 2019;34:259-266
pISSN: 2671-4639 • eISSN: 2671-4663
https://doi.org/10.12750/JARB.34.4.259
JARB
Journal of Animal Reproduction and Biotechnology
Received November 20, 2019
Revised December 3, 2019
Accepted December 11, 2019
*Correspondence
Changhoon Chai
E-mail: chchai@kangwon.ac.kr
ORCID
https://orcid.org/0000-0003-4320-7311
ABSTRACT 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
Copyright © The Korean Society of Animal Reproduction and Biotechnology
CC This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits
unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
J Anim Reprod Biotechnol Vol. 34, No. 4, December 2019
260
sion (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 se-
quencing, 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 tech-
nologies have several issues that remain to be resolved.
For example, emulsion PCR requires the precise opti-
mization 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 compo-
nents and procedures. Technical difficulties associated
with emulsion PCR are discussed, and emulsion PCR-
based technologies are reviewed.
PRINCIPLE OF EMULSION PCR
DNA extracted from a clinical specimen for the diagno-
sis 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, avoid-
ing 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 mo-
nooleate), Span 80 (sorbitan monooleate), Triton X-100
(polyethylene glycol
tert
-octylphenyl ether), ABIL EM 90
(cetyl PEG/PPG-10/1 dimethicone; Evonik Industries,
Essen, Germany), ABIL WE 09 (blend of polyglyceryl-4
isostearate, cetyl PEG/PPG-10/1 dimethicone, and hexyl
laurate; Evonik Industries), and fluorinated silicone are
frequently used as surfactants for emulsion PCR (Williams
et al., 2006; Heredia and Makarewicz, 2016; Witt et al.,
2017). Mineral oil, Tegosoft DEC (diethylhexyl carbon-
ate; Evonik Industries), and silicone oil can be used as the
oil phase of emulsion PCR (Pekin et al., 2010; Heredia
and Makarewicz, 2016; Witt et al., 2017). Surfactants are
generally added to the oil phase. Vortex mixers, magnetic
Polymerase Target DNA Nontarget DNA Primer Surfactant
Oil
Emulsion PCR
Conventional PCR
Target specific PCR
Nonspecific PCR
Fig. 1. Schematic illustrates of conven-
tional PCR and emulsion PCR.
Chai. Emulsion PCR in Biotechnology
261
stirrers, and microfluidic devices can be used to disperse
the aqueous PCR phase in the oil phase (Williams et al.,
2006; Zhu et al., 2012; Kanagal-Shamanna, 2016).
Depending on the specific emulsion PCR method, a
20-200 mL volume of the aqueous PCR phase is added
to the oil phase to prepare a 100-300 mL 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 mm (Shao et al., 2011; Witt et al., 2017).
Droplet diameter and number can be controlled precisely
using microfluidic devices (Byrnes et al., 2018). Microflu-
idic 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
mm (BioRad.).
The 100 mL aqueous PCR phase used for emulsion PCR
may include target and nontarget DNA molecules, 1.25
U polymerase, 0.8 mM (80 pmol per reaction) of forward
and reverse primers, 0.2 mM dNTPs (20 nmol per reac-
tion), and other components (Chai and Oh, 2015; Du et
al., 2019). For a 94 kD polymerase, a typical unit of poly-
merase contains about 8 × 1010 polymerase molecules
(Spangler et al., 2009). Thus, 100 mL 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 (
f
) of aque-
ous droplets containing
k
molecules of individual PCR
components follows the Poisson distribution (Equation 1)
(Swami et al., 2008).
f
(
k
; l) = l
k
·
e
-
l
k
! (Equation 1)
Furthermore, the proportion of the sum of individual
PCR components compartmentalized by
k
units of indi-
vidual PCR components (
N
p
) is given by Equation 2:
P
(
k
; l) =
f
(
k
; l) ·
N
e
·
k
·
1
N
p
=
f
(
k
; l) ·
k
l
(Equation 2)
where l is calculated by dividing
N
p
by the total number
of emulsified aqueous droplets (
N
e
),
k
is the estimated
number of individual PCR components contained in an
emulsified aqueous droplet, and e is the base of the natu-
ral logarithm.
The
P
of polymerase at different
k
values was calcu-
lated using Equation 2 and plotted in Fig. 2A under the
assumption of
N
p
= 1 × 1011 and
N
e
= 109. Given that l is
100, the
P
of polymerase at
k
= 100 is greater than those
of other components (Fig. 2A). The
f
values of polymerase
at
k
values of 46 and 47 are 6.76 × 10-10 and 1.44 × 10-9,
respectively. As
N
e
= 1 × 109, the formation of aqueous
droplets with fewer than 47 polymerases is unlikely. As
seen in the
P
and
f
of polymerase, all aqueous droplets
with
N
e
= 1 × 109 should contain sufficient quantities of
primers and dNTPs for PCR. The
N
e
can be varied from 1
× 105 to 1 × 109 according to the emulsification method
(BioRad.; Diehl et al., 2006; Williams et al., 2006). The
P
of DNA molecules in PCR emulsions is also calculated us-
ing Equation 2. If ≤ 1 × 109 DNA molecules are emulsi-
fied in 1 × 109 aqueous droplets, no aqueous droplet with
more than 11 DNA molecules is likely to be generated (Fig.
2B). The maximum
k
of DNA molecules at
N
e
= 1 × 109
and
N
p
= 1 × 108, 1 × 107, 1 × 106, 1 × 105, 1 × 104, 1
× 103, 1 × 102, and 1 × 101 are 6, 3, 2, 2, 2, 1, 1, and 1,
respectively (Fig. 2B). Assuming that ≤ 105 DNA molecules
are introduced to 105 aqueous droplets, the maximum
k
of DNA molecules in an aqueous droplet at
N
p
= 1 × 105,
1 × 104, 1 × 103, 1 × 102, and 1 × 101 are 8, 3, 2, 1, and
1, respectively (Fig. 2C). Although aqueous droplets in
PCR emulsions may contain more than one DNA molecule
at
N
e
= 1 × 109 and
N
p
= 1 × 108, 1 × 107, 1 × 106, 1 ×
105, and 1 × 104, as well as
N
e
= 1 × 105 and
N
p
= 1 × 105,
1 × 104, and 1 × 103, highly sensitive and specific PCR in
aqueous droplets can be achieved due to the specificity of
PCR primers against their target DNA sequences.
METHODS OF EMULSION PCR
Emulsion PCR is performed via aqueous droplet gen-
eration, 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 mol-
ecules, and conventional PCR is subsequently performed
with collected DNA molecules from the emulsion. Fluo-
rescence-based emulsion PCR uses fluorescent dyes to
J Anim Reprod Biotechnol Vol. 34, No. 4, December 2019
262
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 mL of aqueous PCR phase (containing PCR
components) and 400 mL 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
Cumulative sum of P
B
100
10-1
10-2
10-3
10-4
10-5
10-6
10-7
10-8
10-9
1.0
0.8
0.6
0.4
12345678910 11
1234567891011
Cumulative sum of P
P
k
k
100101102103104
105106107108109
100101102103104
105106107108109
Number of DNAs
k
A
0.05
0.04
0.03
0.02
0.01
0.00
1.0
0.8
0.6
0.2
40 60 80 100 120 140
40 60 80 100 120 140 160
P
0.4
0.0
k
C
10
0
10
-1
10
-2
10
-3
10
-4
10
-5
1.0
0.8
0.6
0.4
12 345678
12 345678
Cumulative sum of P
k
k
P
10
0
10
1
10
2
10
3
10
4
10
5
10
0
10
1
10
2
10
3
10
4
Number of DNAs
10
5
160
Fig. 2. The proportion (
P
) of the sum of Taq polymerase and
DNA molecules compartmentalized by
k
to their total molecule
number (
Np
) based on the Poisson distribution equation. (A)
The
P
of Taq polymerase by
k
(bottom) and the cumulative sum
of
P
(top) under
Np
= 1 × 1011 and number of emulsified aque-
ous droplets in PCR emulsions (
Ne
) = 1 × 109. (B) The
P
of DNA
molecules by
k
(bottom) and cumulative sum of
P
(top) under
Np
= 100 – 109 and
Ne
= 1 × 109. (C) The
P
of DNA molecules by
k
(bottom) and cumulative sum of
P
(top) under
Np
= 1 × 100 -
1 × 105 and
Ne
= 1 × 105.
Chai. Emulsion PCR in Biotechnology
263
et al. used a formulation composed of 150 mL of aque-
ous PCR phase and 600 mL 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 for-
mulas, 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
N
e
for both conventional emulsion PCR meth-
ods is approximately 1 × 109 (Williams et al., 2006; Witt
et al., 2017). The concentration of dNTP in the aqueous
PCR phase for both methods is 0.2 mM (30 and 40 nmol
per 150 and 200 mL PCR reaction, respectively). Under
these conditions, the average
k
of dNTPs is 1.81 - 3.13 ×
107. If a single DNA molecule is compartmentalized in an
aqueous droplet with 1.81 - 3.13 × 107 dNTPs and PCR
is designed to amplify a 500-bp fragment, dNTPs should
be used up after the production of about 3.61 - 6.26 ×
104 DNA amplicons, corresponding to approximately 16
PCR cycles. In addition, 3.61 - 6.26 × 104 DNA amplicons
are not sufficient for identification by electrophoresis;
hence, conventional PCR should be used to amplify en-
riched target DNA molecules after emulsion PCR. DNA in
the emulsion may be extracted by adding organic solvents
(diethyl ether, ethyl acetate, and isobutanol), followed by
aqueous PCR phase recovery by centrifugation and DNA
purification using a commercial kit (Witt et al., 2017).
DNA recovery from PCR emulsions is quite tedious and
complicated. Thus, a simple and easy method of DNA re-
covery from PCR emulsions is required for further devel-
opment of conventional emulsion PCR techniques. Since
they generate a large number (~1 × 109) of aqueous PCR
droplets, conventional emulsion PCR methods are suitable
for enriching low-frequency target DNA molecules in up
to 1 × 109 nontarget DNA molecules (Fig. 2B).
Fluorescence-based emulsion PCR methods include flu-
orescent dyes, such as SYBR green and EvaGreen, or fluo-
rescence 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 mm in diameter that are generated with
20 mL of aqueous PCR phase and 70 mL of oil phase by a
microfluidic droplet generator (BioRad.). The final con-
centration of dNTPs recommended by the droplet digital
PCR system’s manufacturer is 0.8 mM. Under these con-
ditions, the average
k
of dNTPs at
N
e
= 1 × 105 is 9.64 ×
1010. Assuming that the DNA amplicon is 500 bp, the copy
number of DNA amplified with 9.64 × 10 dNTPs may not
exceed 1.93 × 108 and dNTPs may be used up after 28
PCR cycles. However, the manufacturer of the commer-
cial droplet digital PCR system recommends 40 PCR cycles
(BioRad.). Fluorescence-based emulsion PCR methods can
quantify even a single target DNA molecule mixed with
nontarget DNA molecules (Fig. 2C). They may be appro-
priate for early diagnosis of viral and bacterial infection.
Conventional and fluorescence-based emulsion PCR
methods may be performed under regular PCR condi-
tions 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 drop-
lets circulate, collide, and are prone to coalesce in PCR
emulsions (Holtze et al., 2008). The coalescence of aque-
ous PCR droplets may compromise the Poisson distribu-
tion 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-mediat-
ed isothermal DNA amplification in fluorescence-based
emulsion PCR methods confers superior amplification
efficiency and specificity compared with previous meth-
ods (Ma et al., 2018). Moreover, since isothermal DNA
amplification-based methods do not require a thermal
cycler, they may support the development of simple, min-
iaturized fluorescence-based emulsion PCR systems.
APPLICATIONS OF EMULSION PCR IN
BIOTECHNOLOGY
Metagenomic NGS is used to investigate the taxonomic
composition and genetic content of microorganisms in
foods, as well as biological and environmental speci-
mens (Quince et al., 2017; Chiu and Miller, 2019). DNA
J Anim Reprod Biotechnol Vol. 34, No. 4, December 2019
264
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 to-
wards 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 enrich-
ment of DNA or cDNA aptamer candidates during SELEX
(Witt et al., 2017). SELEX is a molecular biological tech-
nique 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 inter-
est is identified. In conventional PCR, low-frequency DNA
or cDNA variants can be lost during enrichment. Enrich-
ment 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 prognos-
tic 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 nor-
mal 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 circulat-
ing tumor DNA assays may be a powerful approach for
evaluating cancer patients’ prognosis and response to
treatment.
CONFLICTS OF INTEREST
No potential conflict of interest relevant to this article
was reported.
ACKNOWLEDGEMENTS
This study was supported by a grant from the National
Research Foundation of Korea (NRF-2013R1A1A2060458),
funded by the Korean government.
AUTHOR’S AFFILIATION, POSITION AND
ORCID NO.
C Chai, Kangwon Nat’l Univ., Assistant Professor,
https://orcid.org/0000-0003-4320-7311
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Book
PREFACE The Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture is involved in agricultural research and development and assists Member States of FAO and IAEA in improving strategies to ensure food security through the use of nuclear techniques and related biotechnologies, where such techniques have a valuable and often unique role. In particular, molecular diagnostic methods have rapidly evolved in the past twenty years, since the advent of the Polymerase Chain Reaction (PCR). They are used in a wide range of agricultural areas such as, improving soil and water management; producing better crop varieties; diagnosing plant and animal diseases; controlling insect pests and improving food quality and safety. The uses of nucleic acid-directed methods have increased significantly in the past five years and have made important contributions to disease control country programmes for improving national and international trade. These developments include the more routine use of PCR as a diagnostic tool in veterinary diagnostic laboratories. However, there are many problems associated with the transfer and particularly, the application of this technology. These include lack of consideration of: the establishment of quality-assured procedures, the required set-up of the laboratory and the proper training of staff. This can lead to a situation where results are not assured. This book gives a comprehensive account of the practical aspects of PCR and strong consideration is given to ensure its optimal use in a laboratory environment. This includes the setting-up of a PCR laboratory; Good Laboratory Practice and standardised of PCR protocols.