Color-coded molecular beacons for multiplex PCR screening assays
Color-coded molecular beacons for multiplex PCR screening assays
Salvatore A. E. Marras 0 1
Sanjay Tyagi 1
Dan-Oscar AntsonID 1
Fred Russell KramerID 0 1
0 Public Health Research Institute, Department of Microbiology , Biochemistry and Molecular Genetics , New Jersey Medical School, Rutgers University , Newark , New Jersey, United States of America, 2 Public Health Research Institute, Department of Medicine, New Jersey Medical School, Rutgers University , Newark , New Jersey, United States of America, 3 Center for Technology Licensing, Weill Cornell Medical Center, Cornell University , New York, New York , United States of America
1 Editor: Ruslan Kalendar, University of Helsinki , FINLAND
The number of different fluorescent colors that can be distinguished in a PCR screening assay restricts the number of different targets that can be detected. If only six colors can be distinguished, and the probe for each target is labeled with a unique color, then only six different targets can be identified. Yet, it is often desirable to identify more targets. For instance, the rapid identification of which bacterial species (if any) is present in a patient's normally sterile blood sample, out of a long list of species, would enable appropriate actions to be taken to prevent sepsis. We realized that the number of different targets that can be identified in a screening assay can be increased significantly by utilizing a unique combination of two colors for the identification of each target species. We prepared a demonstration assay in which 15 different molecular beacon probe pairs were present, each pair specific for the same identifying sequence in the 16S ribosomal RNA gene of a different bacterial species, and each pair labeled with a unique combination of two fluorophores out of the six differently colored fluorophores that our PCR instrument could distinguish. In a set of PCR assays, each containing all 30 color-coded molecular beacons, and each containing DNA from a different bacterial species, the only two colors that arose in each real-time assay identified the species-specific target sequence that was present. Due to the intrinsic low background of molecular beacon probes, these reactions were specific and extremely sensitive, and the threshold cycle reflected the abundance of the target sequence present in each sample.
Data Availability Statement: All relevant data are
within the paper.
Funding: This research was supported by National
Institutes of Health Grant R01 EB000277 and by
National Institutes of Health Grant R01 CA227291.
The open access charge for the publication of this
paper was supported by the laboratory?s (Public
Health Research Institute, Rutgers University)
share of the income received from the
nonexclusive licensing of molecular beacons
technology by PHRI Properties, Inc., which is a
nonprofit corporation wholly owned by Rutgers
When molecular beacon hybridization probes [
] are used to detect different target sequences
in traditional PCR assays, each molecular beacon is labeled with a single uniquely colored
], and the number of different target sequences that can be identified is limited
to the number of differently colored fluorophores that the PCR instrument can distinguish [
]. However, it is often desirable to be able to detect many more target sequences. For example,
University. Neither PHRI Properties, Inc., nor any
of the approximately 75 molecular beacons
licensees, had any role in the study design, data
collection and analysis, decision to publish, or
preparation of the manuscript.
Competing interests: SAEM, ST, and FRK receive
royalties from the licensing of molecular beacons
technology by PHRI Properties, Inc. The relevant
licensed United States patents are: ?Homogeneous
Multiplex Screening Assays and Kits? 7,385,043
and 7,771,949, which were filed in 2003, listing
FRK as the inventor. There are no further United
States patents, products in development, or
marketed products to declare. This does not alter
the authors? adherence to all PLOS ONE policies on
sharing data and materials.
the identification of which species of bacteria is causing an infection, out of a relatively long
list of bacterial species, enables an appropriate antibiotic to be used. We realized that this
limitation can be overcome by utilizing combinations of labeled molecular beacons, in which the
molecular beacon for each target sequence is prepared in two separate portions, and each
portion is labeled with a differently colored fluorophore [
The advantage of such a duplex labeling scheme is that there are more unique combinations
of exactly two colors than the number of colors that can be distinguished. For example, if the
PCR instrument can distinguish six different fluorescent colors, then there are 15 unique
combinations of two colors that can be utilized to label 15 different molecular beacon pairs, each
pair specific for amplicons generated from a different target. As a result of this labeling scheme,
one portion of the amplicons arising from a particular target sequence are labeled in one color
and a second portion of the amplicons arising from the same target sequence are labeled in a
different color. The combination of colors labeling the amplicons generated from a particular
target sequence in the sample being tested serves as a code that unambiguously identifies the
target that is present. This labeling paradigm is particularly useful for homogeneous, real-time
PCR screening assays in which only one target sequence is likely to be present in the sample.
We prepared a demonstration assay, rather than an actual clinical assay, to illustrate the
specificity and sensitivity of utilizing color-coded molecular beacons in multiplex, real-time
PCR screening assays. This assay was designed to rapidly detect, identify, and quantitate 15
different bacterial species. The presence of any one of them in a normally sterile blood sample
would indicate that sepsis could occur [
]. A pair of PCR primers that are specific for
conserved sequences within the V3 region of the 16S ribosomal RNA gene of all 15 bacterial
species was used to amplify the target sequences [
]. In addition, 15 pairs of color-coded
molecular beacons were present in the PCR assay mixture (30 different probes in all), each
pair specific for a unique variable sequence that is present in the amplified DNA from one of
the 15 bacterial species, and each pair labeled with a unique combination of two differently
colored fluorophores out of the six differently colored fluorophores that the PCR instrument
could distinguish. The results confirm that color-coded molecular beacon probes, by virtue of
their hairpin structure can distinguish targets that differ by as little as a single-nucleotide
polymorphism, and when they are not hybridized to their target, their fluorescence is quenched to
such a great extent that many different color-coded molecular beacons can be present in a
PCR assay without compromising the sensitivity of the assay.
Cultures of 16 different sepsis-causing bacterial species were purchased from the American
Type Culture Collection (Manassas, VA). The genomic DNA from each species was isolated
and used as a template for the generation of copies of the V3 region of its 16S ribosomal RNA
gene, utilizing the following PCR primers: 5?-ACTCCTACGGGAGGCAGCAGT-3? and
5?TATTACCGCGGCTGCTGGCAC-3? (Integrated DNA Technologies, Coralville, IA). Each
blunt-ended amplicon was cloned into the Srf I restriction site of a pPCR Script Amp SK(+)
plasmid, utilizing a PCR-Script Amp Cloning Kit (Stratagene, La Jolla, CA). Target plasmids
for use in the PCR screening assays were isolated from the transformed cells utilizing a
CompactPrep Plasmid Kit (Qiagen, Hilden, Germany), and the plasmids were linearized by
digestion with either Kpn I or Cla I restriction endonuclease, and their concentration was
determined in a NanoDrop Spectrophotometer (ThermoFisher Scientific, Waltham, MA). The
nucleotide sequence of the single bacterial gene segment inserted into each plasmid was
confirmed by automated Sanger sequence analysis. These 16 linearized plasmids were used as
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targets for the demonstration assays (one of which served as a negative control to assess
Color-coded molecular beacons
Fifteen pairs of molecular beacon probes were synthesized, each pair specific for a unique
target sequence present in a different bacterial species (Table 1). For designing the molecular
beacons, the mfold Web Server was used to predict stem melting temperatures and to check
that no internal stem-loop structures will form (http://unafold.rna.albany.edu/?q=mfold/
DNA-Folding-Form); and the OligoAnalyzer 3.1 computer program was used to predict
probe-target hybrid melting temperatures (http://www.idtdna.com/calc/analyzer). The
molecular beacons were synthesized in our laboratory on a 394 DNA/RNA synthesizer (Applied
Biosystems, Foster City, CA) utilizing ?-cyanoethyl phosphoramidite precursors (Glen Research,
Sterling, VA) on either Dabcyl controlled-pore glass columns (Glen Research) or on Black
Hole Quencher-2 (BHQ-2) controlled-pore glass columns (LGC Biosearch Technologies,
Petaluma, CA). The addition of fluorescein (FAM) and tetrachlorofluorescein (TET) was
accomplished through the use of fluorophore-modified phosphoramidites; and the addition of other
fluorophores was accomplished by incorporating an amino-modifier phosphoramidite at the
5?-terminal position of each oligonucleotide and then reacting those amino groups overnight
in 100 mM NaHCO3 at 37?C with a succinimidyl ester derivative of one of the following
fluorophores: Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594 (all from ThermoFisher Scientific),
or Atto 532 (from Sigma-Aldrich, St. Louis, MO). Each molecular beacon was purified on a
System Gold High Performance Liquid Chromatograph (Beckman Coulter, Brea, CA) through
a C-18 reverse-phase column (Waters Corporation, Milford, MA). A detailed protocol for
molecular beacon synthesis is available at http://www.molecular-beacons.org.
Polymerase chain reactions
Each 75-?L PCR assay contained 50 mM KCl, 6 mM MgCl2, 10 mM Tris-HCl (pH 8.0),
250 ?M dATP, 250 ?M dCTP, 250 ?M dGTP, 250 ?M dTTP, 3.75 Units of AmpliTaq Gold
DNA polymerase (ThermoFisher Scientific), 50 nM limiting primer and 1,000 nM excess
primer (Integrated DNA Technologies) whose nucleotide sequences are described below,
100,000 linearized plasmid DNA target molecules, and a 2,062 nM mixture of 30 different
color-coded molecular beacons. The reactions were carried out in 200-?L white polypropylene
tubes (USA Scientific, Ocala, FL) on an Applied Biosystems PRISM 7700 real-time
spectrofluorometric thermal cycler, utilizing its Sequence Detector Version 1.6.3 analysis program to
deconvolve the emission spectra. The reactions were incubated for 10 min at 95?C to activate
the polymerase, followed by 40 cycles of 95?C denaturation for 15 sec, 58?C annealing for 30
sec, and 72?C chain elongation for 20 sec. The fluorescence intensity of each of the six
fluorophores was measured during the 58?C annealing stage of each thermal cycle. The length of
the PCR amplicons generated from each of the 16 different bacterial target plasmids varied
between 162 and 188 base pairs.
Design of the assays
Selection of the target sequences. The 16S ribosomal RNA gene of bacteria is a useful
PCR target sequence for the identification of bacterial species because it includes sequence
regions that are highly conserved from species to species, enabling a single pair of primers to
be used for the generation of amplicons. These conserved DNA sequences encode portions of
the ribosomal RNA to which ribosomal proteins bind. However, the sequences that lie between
these conserved sites vary from species to species, providing an ?identification bracelet? within
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There are 15 unique combinations of six differently colored fluorophore labels. Emission maxima: FAM = 512 nm; TET = 536 nm; Atto 532 = 553 nm; Alexa Fluor
546 = 572 nm; Alexa Fluor 568 = 603 nm; Alexa Fluor 594 = 618 nm. The complementary arm sequences of the molecular beacons are underlined. In some probe pairs,
the probe sequences are slightly different in length to assure similar melting temperatures. Also, to assure similar detection intensities, the concentration of each
molecular beacon was adjusted, with the actual concentration being equal to the relative concentration x 13.3 nM.
the amplicons that can be read by color-coded molecular beacon hybridization probes. We
therefore designed a PCR assay that could distinguish 15 different bacterial species by virtue of
the unique sequence present in the V3 region of their 16S ribosomal RNA gene. Plasmids were
prepared that contained the 16S ribosomal RNA gene target sequence for each of the 15
bacterial species. Table 2 shows the PCR priming sites and the unique molecular beacon target
sequence that is present within each target plasmid.
Design of the PCR primers and their non-symmetric concentrations
Two PCR primers were prepared: a ?limiting primer? that was present in the assays at 50 nM,
and an ?excess primer? that was present in the assays at 1,000 nM. The purpose of utilizing
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Underlined nucleotides in the limiting primer target sequence differ from species to species, but form a base pair with the corresponding inosine in the limiting primer.
The Streptococcus pyogenes plasmid served as a control in a screening assay that contained all 30 molecular beacon probes, but did not contain any molecular beacon
probes for Streptococcus pyogenes.
non-symmetric primer concentrations is that both primers take part in the initial exponential
amplification of the target sequence, but after the limiting primer is used up, the excess primer
continues the linear amplification of one of the two amplicon strands, thereby providing
single-stranded targets for the hybridization of the molecular beacons, with virtually no
competition from the complementary amplicon strand [
In order to enhance the specificity of priming, and to minimize the possibility of
primerdimer formation, each primer was designed to form a hairpin structure in which the
nucleotide sequence of its 3? arm, as well as all or part of the sequence in its loop, was complementary
to its priming site in the target DNA sequence [
]. The sequence of the excess primer was
5?-GTGCCTTACCGCGGCTGCTGGCAC-3?, where the underlined nucleotides form the
hairpin stem; and the entire loop sequence and the 3?-arm sequence were complementary to the
conserved priming site in every bacterial target DNA. The sequence of the limiting primer was
5?-ATTCCCggggccIGAGGCAGCAGTIGGGAAT-3?, where the underlined nucleotides
form the hairpin stem; but in this primer only the capitalized nucleotides in the loop sequence
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and the nucleotides in the 3?-arm sequence were complementary to the priming site in the
bacterial target DNAs. Also, the limiting primer contained two inosine nucleotides (?I?), which
can form a base pair with any of the four common nucleotides [
], as the complementary
nucleotide in the corresponding position in the priming site of each bacterial species
sometimes varied (see Table 2). The additional nucleotides in the loop of the limiting primer
(represented by lower case letters) were present to assure that the limiting primer formed a strong
hybrid with the amplicons, despite the lowering concentration of the limiting primer as it was
used up during exponential synthesis.
Design of the color-coded molecular beacon probes
Fifteen pairs of color-coded molecular beacons were designed for use in the PCR screening
assays (Table 1). The nucleotide sequence in the loop of each pair of molecular beacons was
perfectly complementary to the unique target sequence present in the amplicons synthesized
from that pair?s intended bacterial target sequence (Table 2). Each pair of molecular beacons
was labeled with two uniquely colored fluorophores out of the six differently colored
fluorophores that could be distinguished by the PCR instrument. In designing the molecular beacon
set, in order to ensure specificity, our goal was to utilize molecular beacons that would
hybridize to their targets and generate a fluorescence signal only if their probe sequence was perfectly
complementary to their intended target. If the probe sequence in the loop was not
complementary to an available target sequence within an amplicon, the molecular beacon was designed to
retain its hairpin shape, to not bind to that target, and to remain dark.
The high specificity of molecular beacon probes that distinguishes them from conventional
linear hybridization probes is due to their hairpin shape [
]. When a molecular beacon is
hybridized to its target sequence, it forms a relatively rigid probe-target double helix [
prevents the arms of the hairpin from interacting with each other, separating the fluorophore
from the quencher, resulting in a bright fluorescence signal in the characteristic color of the
fluorophore. However, if there is a mismatch between the sequence in the loop of the
molecular beacon and its target sequence, then the molecular beacon is more stable retaining its
hairpin shape, in which its fluorescence is quenched by contact between the fluorophore and the
quencher. As a consequence, molecular beacons can be designed so that they only form a
probe-target hybrid if every nucleotide in the loop sequence is complementary to the
corresponding nucleotide in the target sequence [
In particular, to ensure the high specificity of each of the 30 different molecular beacons
included in the demonstration assay, the length of the probe sequence in the loop of the
hairpin, and the length of the complementary arm sequences in the hairpin stem, were designed
to exhibit a very similar stability under the conditions present during the annealing stage of
each thermal cycle (which is the stage during which molecular beacon fluorescence was
measured). Moreover, since the stability of the hairpin stem of a molecular beacon depends on the
strength of the transient bonds formed between the fluorophore and the quencher [
], as well
as the strength of the nucleotide pairs in the stem helix, molecular beacons possessing a
BHQ2 quencher (which forms strong bonds with fluorophores) only required a stem that was five
base pairs long, whereas the stems of the molecular beacons possessing a Dabcyl quencher
needed to be six base pairs long.
Adjusting the fluorescence intensities of the molecular beacons in the set
The fluorescence intensity of each of the six fluorophores at the PCR annealing temperature is
dependent on the nature and intensity of the light used to excite their fluorescence. We carried out
our PCR screening assays in an Applied Biosystems PRISM 7700 real-time spectrofluorometric
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thermal cycler, because this instrument can distinguish six different fluorescent colors. However,
in this instrument fluorescence is excited by a blue argon ion laser, which efficiently stimulates
green and yellow fluorescence in fluorophores such as FAM, TET, Atto 532, and Alexa Fluor
546, but is less efficient at stimulating orange and red fluorescence in fluorophores such as Alexa
Fluor 568 and Alexa Fluor 594. Consequently, in preparing the mixture of molecular beacons to
use in the demonstration PCR screening assays, we adjusted the concentration of each of the 30
different molecular beacons, utilizing more of those molecular beacons whose fluorophore is
inefficiently stimulated by the blue laser and less of those molecular beacons whose fluorophore
is more efficiently stimulated. The goal was to create a mixture in which the fluorescence
intensity of the two molecular beacons in each pair was approximately the same. Table 1 lists the
resulting concentration of each molecular beacon that was present in the PCR screening assays.
PCR screening assays
Sixteen PCR screening assays were carried out, each initiated with 100,000 linearized target
plasmids possessing the V3 region of the 16S ribosomal RNA gene from a different bacterial
species. Each assay contained 50 nM of the limiting primer, 1,000 nM of the excess primer,
and a mixture of 15 pairs of concentration-adjusted color-coded molecular beacons, each pair
specific for the amplicons generated by a different bacterial species. One assay, containing
target plasmids for Streptococcus pyogenes, served as an illustrative negative control, as there were
no molecular beacons in the assay mixture that were specific for that bacterial species.
Fluorescence was read during the annealing stage of each thermal cycle. The results are shown in Fig
1. Only two unique fluorescent colors (out of the six colors that the PCR instrument could
distinguish) arose in each assay, and the particular combination of two colors uniquely identified
each target species. No colors arose in the control assay, emphasizing the low background and
excellent specificity of color-coded molecular beacon probes.
Quantitation and sensitivity
Six PCR assays were carried out, each initially containing a different quantity of Staphylococcus
aureus targets (either 0; 10; 100; 1,000; 10,000; or 100,000 linearized plasmids), and each
containing all 15 pairs of color-coded molecular beacons. The results are shown in Fig 2.
Irrespective of whether there was initially a large number of targets or a small number of targets, only
two of the six fluorescent colors arose, and they were the two colors that identified the targets
as containing a sequence from Staphylococcus aureus. The fewer the number of targets initially
present in the sample, the more thermal cycles were needed to be completed before the
fluorescence signal arose above background (the threshold cycle). These results were in line with the
classic relationship governing exponential amplification assays, in which the time it takes to
generate a signal is inversely linearly proportional to the logarithm of the number of targets
originally present in the sample [
]. Most significantly, there was a clear signal from only
ten target molecules, indicating that these real-time PCR screening assays, which utilize a
mixture of 30 different color-coded molecular beacons, are as sensitive as traditional PCR assays
that only possess a few probes.
Intensity adjusted, uniquely color-coded molecular beacons
The PCR screening assay that we designed was a demonstration assay for the rapid
identification of 15 different bacterial species that can cause sepsis, rather than an actual clinical assay,
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Fig 1. Screening assays. Real-time PCR assays containing 30 different color-coded molecular beacons were carried out, each assay initiated with
100,000 linearized target plasmids that contained a copy of the V3 region of the 16S ribosomal RNA gene from a different bacterial species. The unique
duplex color combination that arose in each of the 15 screening assays unambiguously identified the species-specific target sequence that was initially
present in each sample. A control assay containing a target plasmid from Streptococcus pyogenes, for which there were no molecular beacon probes, gave
a very low background signal in all six colors, and remained low throughout the course of the assay.
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Fig 2. Sensitivity confirmation. Real-time PCR screening assays containing all 30 color-coded molecular beacons were initiated with different
numbers of copies of the Staphylococcus aureus target plasmid. Fluorescent signals only arose from the two species-specific molecular beacons labeled
with FAM and Alexa Fluor 568. The two-color threshold cycle of each reaction was inversely linearly proportional to the logarithm of the number of
target molecules initially present in each sample. Low background signals from the 28 non-hybridized molecular beacons enabled the detection of as
few as ten target molecules.
which would require extensive validation with clinical samples. Such a clinical assay would
rapidly identify the species that is present in a blood sample (if any) in order to administer an
effective antibiotic. Current practice is to send the blood sample out for culture (which takes a
day or two) and to immediately administer a broad spectrum antibiotic (which may not be
needed, and potentially could enable the growth of antibiotic-resistant strains throughout
the body). Moreover, if the patient is infected, the broad spectrum antibiotic that is
administered might be ineffective against the actual species that is present, and the patient could die of
Our assay was designed to demonstrate the advantages of using uniquely color-coded
molecular beacons in real-time PCR screening assays, enabling both the identity and the
abundance of the target sequence to be determined. The resulting signal for each target is composed
of a unique combination of exactly two colors. Moreover, the intensity of each member of the
pair of identified colors is approximately equal, because the concentration of each differently
labeled portion was adjusted by taking into consideration the intrinsic intensity of each
fluorophore in the PCR buffer at the annealing temperature, and the efficiency of detection of each
fluorophore by the particular PCR instrument in which the screening assay was carried out.
Furthermore, the results demonstrate that the use of hairpin-shaped molecular beacon probes
assures that background fluorescence is extremely low, enabling sensitive detection of the
We purposely utilized the same number of colors (two) to label the molecular beacons for
each target in our assay. The advantage of using only molecular beacons that are divided into
the same number of portions is that the expected outcome is a signal composed of the same
number of different colors (or the absence of a signal, should no target be present in the
sample). Consequently, if more than the expected number of colors occur, that result would
indicate that two or more different targets are present in the sample. However, when utilizing two
colors for each target, if the sample unexpectedly contains two different targets whose identity
is ambiguous, and the resulting signal consists of three or four colors, their identities can be
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unambiguously resolved by running a second assay with the same sample that utilizes a
different set of two colors for each of the 15 pairs of molecular beacon probes. With this alternative
labeling scheme, the result will be a different set of three or four colors, which compared to the
original set of three or four colors enables the identification of the species involved [
The results suggest that higher multiplicities can be achieved. Using an instrument that can
distinguish six different fluorescent colors, and if three different molecular beacon probes are
utilized for each target, and if each of these three probes bears a differently colored
fluorophore, then the occurrence of a signal containing that unique combination of three colors will
indicate that the target of that probe set is present in the sample. Since there are 20 different
unique combinations of three colors that can be assembled from the six colors that the
instrument can distinguish, the screening assay will be able to identify 20 different target sequences.
Although this triplex-coding arrangement would require 60 different molecular beacons, our
results suggest that there should be no loss of sensitivity if they are well-designed. Moreover, if
the PCR instrument can distinguish seven different fluorescent colors, then the number of
targets that can be distinguished when three differently colored molecular beacons are used to
identify each target is 35.
Earlier designs for enhancing the multiplicity of screening assays that employ
combinatorially labeled hybridization probes that intermix singly labeled probes with probes labeled in
different numbers of colored portions (and whose signal intensities were not adjusted) [
generate results that are more difficult to interpret, especially if multiple targets are present in
the sample. Alternative schemes for enhancing the multiplicity of detection, that employ
combinations of melting temperatures from differently colored probe-target hybrids to identify the
], are carried out at end-point and are not able to determine the concentration of
the target in the sample.
Detecting each target with multiple molecular beacons in these multiplex assays may
increase the cost of assembling the assay for the first time, however, if the assay is used over
and over, this increased expenditure will become a minor fraction of the overall cost.
Furthermore, since each sample will be assayed just once, rather than multiple times, the overall cost
will be less.
Multiplex digital PCR applications
Perhaps the most exciting application for adjusted, uniquely color-coded molecular beacons
that are designed for use in screening assays, involves their inclusion in assays that are carried
out on digital PCR instruments. In digital PCR assays, the entire PCR mixture, including the
sample and the hybridization probes, is divided into a very large number of individual wells or
droplets (sometimes more than a million picoliter-sized droplets), with the result that only one
target molecule (or no target molecule) is present in each droplet; and then simultaneous PCR
is carried out in all of the droplets, and the amplicons that are generated in each droplet are
then identified by virtue of the colored fluorescence signals that arise [
]. Therefore, each
droplet is a PCR screening assay that identifies the one target molecule (if any) that is present
in that droplet. Moreover, and this is a key point, although the PCR assay carried out in each
droplet is a screening assay for the one target molecule that may be initially present in the
droplet, the overall digital PCR assay is multiplex, able to identify and quantitate all of the
different targets that are present in a sample (the abundance of each being determined by the
number of droplets that are lit up in the same unique color combination).
There is great interest in utilizing digital PCR to simultaneously detect and quantitate a
number of different targets in a sample [
]. However, the use of digital PCR for multiplex
assays has been limited, because currently available digital PCR instruments can only
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distinguish two or three different fluorescent colors [
]. However, that will soon change.
Digital PCR instruments are being developed that can distinguish as many as six different
fluorescent colors [
]. Once this occurs, it should be very exciting to include uniquely color-coded
molecular beacons in the digital PCR assay mixtures for the simultaneous identification and
quantitation of 15 or 20 different targets in a sample. These digital PCR assays should be
sensitive, quantitative, and highly reliable.
The authors are grateful to Diana Vargas-Gold for thoughtful discussions and critical review
of the manuscript.
Conceptualization: Salvatore A. E. Marras, Sanjay Tyagi, Dan-Oscar Antson, Fred Russell
Formal analysis: Salvatore A. E. Marras, Sanjay Tyagi, Dan-Oscar Antson, Fred Russell
Funding acquisition: Sanjay Tyagi, Fred Russell Kramer.
Investigation: Salvatore A. E. Marras, Dan-Oscar Antson.
Methodology: Salvatore A. E. Marras, Sanjay Tyagi, Dan-Oscar Antson, Fred Russell Kramer.
Resources: Salvatore A. E. Marras.
Visualization: Salvatore A. E. Marras.
Writing ? original draft: Fred Russell Kramer.
Writing ? review & editing: Fred Russell Kramer.
11 / 12
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