Name : Diana Mohammed Rafie

National University for Science and Technology Subject : Molecular Biology

Deanship of the College of Medical Stage : Second
and Health Technologies Date :12/Mar /2020
The Report (2019-2020)
Report Title : Digital polymerase chain reaction

igital polymerase chain reaction (digital PCR, Digital PCR, 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] 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

Principles:

The polymerase chain reaction method is used to quantify nucleic acids by amplifying a
nucleic acid molecule with the enzyme DNA polymerase.[2] 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.

Figure 1. Oil droplets containing fluorescent PCR target molecule

 Name : Diana Mohammed Rafie
National University for Science and Technology Subject : Molecular Biology
Deanship of the College of Medical Stage : Second
and Health Technologies Date :12/Mar /2020
The Report (2019-2020)
Report Title : Digital polymerase chain reaction

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

Instead of performing one reaction per well, dPCR involves partitioning the PCR solution
into tens of thousands of nano-liter sized droplets, where a separate PCR reaction takes place
in each one.[3][4] A PCR solution is made similarly to a TaqMan assay, which consists of
template DNA (or RNA), fluorescence-quencher probes, primers, and a PCR master mix,
which contains DNA polymerase, dNTPs, MgCl2, and reaction buffers at optimal
concentrations. Several different methods can be used to partition samples, including
microwell plates, capillaries, oil emulsion, and arrays of miniaturized chambers with nucleic
acid binding surfaces.[5] 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 readout of “0” or “1”. The fraction of fluorescing droplets is
recorded.[4] 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 droplet. 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.
[6]
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. Different from many peoples's belief that dPCR provides absolute
quantification, digital PCR uses statistical power to provide relative quantification. For
example, if Sample A, when assayed in 1 million partitions, gives one positive reaction, it
does not mean that the Sample A has one starting molecule.

The benefits of dPCR include increased precision through massive sample partitioning, which
ensures reliable measurements in the desired DNA sequence due to reproducibility.[4] Error
rates are larger when detecting small-fold change differences with basic PCR, while error
rates are smaller with dPCR due to the smaller-fold change differences that can be detected in
DNA sequence. The technique itself reduces the use of a larger volume of reagent needed,
 Name : Diana Mohammed Rafie
National University for Science and Technology Subject : Molecular Biology
Deanship of the College of Medical Stage : Second
and Health Technologies Date :12/Mar /2020
The Report (2019-2020)
Report Title : Digital polymerase chain reaction

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.

Comparison between 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 indicates the
initial amount of template nucleic acid. Similarly, qPCR utilizes fluorescence; however, it
measures the intensity of fluorescence at specific times (generally after every amplification
cycle) to determine the relative amount of target molecule (DNA), but cannot specify the
exact amount without constructing a standard curve using different amounts of a defined
standard. 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 may not
be as precise due to the extrapolation required to attain a measurement.[5][7]

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 may theoretically
affect results due to the stopping of the experiment In practice, however, most qPCR thermal
cyclers read each sample's fluorescence very quickly at the end of the annealing/extension
step before proceeding to the next melting step, meaning this hypothetical concern is not
actually relevant or applicable for the vast majority of researchers.

qPCR is unable to distinguish differences in gene expression or copy number variations 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%.[9]

Applications:

Digital PCR has many applications in basic research, clinical diagnostics and environmental
testing. Its uses include pathogen detection and digestive health analysis; liquid biopsy for
cancer monitoring, organ transplant rejection monitoring and non-invasive prenatal testing
for serious genetic abnormalities copy number variation analysis, single gene expression
analysis, rare sequence detection, gene expression profiling and single-cell analysis; the
 Name : Diana Mohammed Rafie
National University for Science and Technology Subject : Molecular Biology
Deanship of the College of Medical Stage : Second
and Health Technologies Date :12/Mar /2020
The Report (2019-2020)
Report Title : Digital polymerase chain reaction

detection of DNA contaminants in bioprocessing, the validation of gene edits and detection
of specific methylation changes in DNA as biomarkers of cancer. dPCR is also frequently
used as an orthogonal method to confirm rare mutations detected through next-generation
sequencing (NGS) and to validate NGS libraries.

Absolute quantification:

dPCR enables the absolute and reproducible quantification of target nucleic acids at single-
molecule resolution. Unlike analogue quantitative PCR (qPCR), however, absolute
quantification with dPCR does not require a standard curve).[40] dPCR also has a greater
tolerance for inhibitor substances and PCR assays that amplify inefficiently as compared to
qPCR.

dPCR can quantify, for example, the presence of specific sequences from contaminating
genetically modified organisms in foodstuffs,[45] viral load in the blood,[46] PBMCs,[47][48]
serum samples,[49] chorionic villi tissues,[50][51] biomarkers of neurodegenerative disease in
cerebral spinal fluid,[52] and fecal contamination in drinking water. [53]

Copy number variation:

An alteration in copy number state with respect to a single-copy reference locus is referred to
as a “copy number variation” (CNV) if it appears in germline cells, or a copy number
alteration (CNA) if it appears in somatic cells. A CNV or CNA could be due to a deletion or
amplification of a locus with respect to the number of copies of the reference locus present in
the cell, and together, they are major contributors to variability in the human genome.[55][56][57]
They have been associated with cancers; neurological, psychiatric, and autoimmune diseases;
and adverse drug reactions. However, it is difficult to measure these allelic variations with
high precision using other methods such as qPCR, thus making phenotypic and disease
associations with altered CNV status challenging.

The large number of “digitized,” endpoint measurements made possible by sample

partitioning enables dPCR to resolve small differences in copy number with better accuracy
and precision when compared to other methods such as SNP-based microarrays[68] or qPCR.
qPCR is limited in its ability to precisely quantify gene amplifications in several diseases,
including Crohn’s disease, HIV-1 infection, and obesity.

dPCR was designed to measure the concentration of a nucleic acid target in copies per unit
volume of the sample. When operating in dilute reactions where less than ~10% of the
partitions contain a desired target (referred to as “limiting dilution”), copy number can be
estimated by comparing the number of fluorescent droplets arising from a target CNV with
the number of fluorescent droplets arising from an invariant single-copy reference locus.[20] In
fact, both at these lower target concentrations and at higher ones where multiple copies of the
 Name : Diana Mohammed Rafie
National University for Science and Technology Subject : Molecular Biology
Deanship of the College of Medical Stage : Second
and Health Technologies Date :12/Mar /2020
The Report (2019-2020)
Report Title : Digital polymerase chain reaction

same target can co-localize to a single partition, Poisson statistics are used to correct for these
multiple occupancies to give a more accurate value for each target’s concentration.

Digital PCR has been used to uncover both germline and somatic variation in gene copy
number between humans and to study the link between amplification of HER2 (ERBB2) and
breast cancer progression.

Rare mutation and rare allele detection:

Partitioning in digital PCR increases sensitivity and allows for detection of rare events,
especially single nucleotide variants (SNVs), by isolating or greatly diminishing the target
biomarker signal from potentially competing background. These events can be organized into
two classes: rare mutation detection and rare sequence detection.

Rare Mutation Detection:

Rare mutation detection occurs when a biomarker exists within a background of a highly
abundant counterpart that differs by only a single nucleotide variant (SNV). Digital PCR has
been shown to be capable of detecting mutant DNA in the presence of a 200,000-fold excess
of wild type background, which is 2,000 times more sensitive than achievable with
conventional qPCR.[7]

Rare Sequence Detection:

Digital PCR can detect rare sequences such as HIV DNA in patients with HIV, and DNA
from fecal bacteria in ocean and other water samples for assessing water quality. dPCR can
detect sequences as rare as 1 in every 1,250,000 cells.[19]

Liquid Biopsy:

dPCR’s ability to detect rare mutations may be of particular benefit in the clinic through the
use of the liquid biopsy, a generally noninvasive strategy for detecting and monitoring
disease via bodily fluids. Researchers have used liquid biopsy to monitor tumor load,
treatment response and disease progression in cancer patients by measuring rare mutations in
circulating tumor DNA (ctDNA) in a variety of biological fluids from patients including
blood, urine and cerebrospinal fluid.. Early detection of ctDNA (as in molecular relapse) may
lead to earlier administration of an immunotherapy or a targeted therapy specific for the
patient’s mutation signature, potentially improving chances of the treatment’s effectiveness
rather than waiting for clinical relapse before altering treatment. Liquid biopsies can have
turnaround times of a few days, compared to two to four weeks or longer for tissue-based
tests. This reduced time to results has been used by physicians to expedite treatments tailored
to biopsy data.
 Name : Diana Mohammed Rafie
National University for Science and Technology Subject : Molecular Biology
Deanship of the College of Medical Stage : Second
and Health Technologies Date :12/Mar /2020
The Report (2019-2020)
Report Title : Digital polymerase chain reaction

In 2016, a prospective trial using dPCR at the Dana-Farber Cancer Institute authenticated the
clinical benefit of liquid biopsy as a predictive diagnostic tool for patients with non-small-cell
lung cancer. The application of liquid biopsy tests have also been studied in patients with
breast, colorectal, gynecologic, and bladder cancers to monitor both the disease load and the
tumor’s response to treatment.

Gene expression and RNA quantification:

Gene expression and RNA quantification studies have benefited from the increased precision
and absolute quantification of dPCR. RNA quantification can be accomplished via RT-PCR,
wherein RNA is reverse-transcribed into cDNA in the partitioned reaction itself, and the
number of RNA molecules originating from each transcript (or allelic transcript) is quantified
via dPCR (ref).[26]

One can often achieve greater sensitivity and precision by using dPCR rather than qPCR to
quantify RNA molecules in part because it does not require use of a standard curve for
quantification.[90] dPCR is also more resilient to PCR inhibitors for the quantification of RNA
than qPCR.[43][11]

dPCR can detect and quantify more individual target species per detection channel than
qPCR by virtue of being able to distinguish targets based on their differential fluorescence
amplitude or by the use of distinctive color combinations for their detection.[91] As an
example of this, a 2-channel dPCR system has been used to detect in a single well the
expression of four different splice variants of human telomerase reverse transcriptase, a
protein that is more active in most tumor cells than in healthy cells.

Alternative Uses for Partitioning:

Using the dynamic partitioning capabilities employed in dPCR, improved NGS sequencing
can be achieved by partitioning of complex PCR reactions prior to amplification to give more
uniform amplification across many distinct amplicons for NGS analysis. Additionally, the
improved specificity of complex PCR amplification reactions in droplets has been shown to
greatly reduce the number of iterations required to select for high affinity aptamers in the
SELEX method. Partitioning can also allow for more robust measurements of telomerase
activity from cell lysates. dPCR’s dynamic partitioning capabilities can also be used to
partition thousands of nuclei or whole cells into individual droplets to facilitate library
preparation for a single cell assay for transposase-accessible chromatin using
sequencing (scATAC-seq).[98]
 Name : Diana Mohammed Rafie
National University for Science and Technology Subject : Molecular Biology
Deanship of the College of Medical Stage : Second
and Health Technologies Date :12/Mar /2020
The Report (2019-2020)
Report Title : Digital polymerase chain reaction

Droplet Digital PCR:

Droplet Digital PCR (ddPCR) is a method of dPCR in which a 20 microliter sample reaction
including assay primers and either Taqman probes or an intercalating dye, is divided into
~20,000 nanoliter-sized oil droplets through a water-oil emulsion technique, thermocycled to
endpoint in a 96-well PCR plate, and fluorescence amplitude read for all droplets in each
sample well in a droplet flow cytometer.