Digital PCR
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Digital polymerase chain reaction (digital PCR, DigitalPCR, dPCR, or dePCR) is a
biotechnological Biotechnology is the integration of natural sciences and engineering sciences in order to achieve the application of organisms, cells, parts thereof and molecular analogues for products and services. The term ''biotechnology'' was first used by ...
refinement of conventional
polymerase chain reaction The polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies (complete or partial) of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it (or a part of it) t ...
methods that can be used to directly quantify and clonally amplify nucleic acids strands including DNA,
cDNA In genetics, complementary DNA (cDNA) is DNA synthesized from a single-stranded RNA (e.g., messenger RNA (mRNA) or microRNA (miRNA)) template in a reaction catalyzed by the enzyme reverse transcriptase. cDNA is often used to express a speci ...
, or
RNA Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. RNA and deoxyribonucleic acid ( DNA) are nucleic acids. Along with lipids, proteins, and carbohydra ...
. 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. 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 Massive parallel sequencing or massively parallel sequencing is any of several high-throughput approaches to DNA sequencing using the concept of massively parallel processing; it is also called next-generation sequencing (NGS) or second-generation s ...
.


Principles

The polymerase chain reaction method is used to quantify
nucleic acids Nucleic acids are biopolymers, macromolecules, essential to all known forms of life. They are composed of nucleotides, which are the monomers made of three components: a 5-carbon sugar, a phosphate group and a nitrogenous base. The two main clas ...
by amplifying a nucleic acid molecule with the enzyme
DNA polymerase A DNA polymerase is a member of a family of enzymes that catalyze the synthesis of DNA molecules from nucleoside triphosphates, the molecular precursors of DNA. These enzymes are essential for DNA replication and usually work in groups to create ...
. 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. 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. A PCR solution is made similarly to a
TaqMan TaqMan probes are hydrolysis probes that are designed to increase the specificity of quantitative PCR. The method was first reported in 1991 by researcher Kary Mullis at Cetus Corporation, and the technology was subsequently developed by Hoffmann-L ...
assay, which consists of template DNA (or RNA), fluorescence-quencher probes, primers, and a PCR
master mix ''Master Mix'' is a remix album by Australian synthpop band Real Life. The album was released in a limited edition in Australia in November 1984 and features remixed versions of songs from the band's debut studio album ''Heartland''. The album p ...
, which contains
DNA polymerase A DNA polymerase is a member of a family of enzymes that catalyze the synthesis of DNA molecules from nucleoside triphosphates, the molecular precursors of DNA. These enzymes are essential for DNA replication and usually work in groups to create ...
, 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. 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. The partitioning of the sample allows one to estimate the number of different molecules by assuming that the molecule population follows the
Poisson distribution In probability theory and statistics, the Poisson distribution is a discrete probability distribution that expresses the probability of a given number of events occurring in a fixed interval of time or space if these events occur with a known co ...
, 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. 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 people'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. 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, 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 A real-time polymerase chain reaction (real-time PCR, or qPCR) is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR (i.e., in real ...
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. 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 cycler The thermal cycler (also known as a thermocycler, PCR machine or DNA amplifier) is a laboratory apparatus most commonly used to amplify segments of DNA via the polymerase chain reaction (PCR). Thermal cyclers may also be used in laboratories to fa ...
s 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. dPCR measures the amplification by measuring the products of end point PCR cycling and is therefore less susceptible to the artifacts arising from impaired amplification efficiencies due to the presence of PCR inhibitors or primer template mismatch. qPCR is unable to distinguish differences in gene expression or copy number variations that are smaller than twofold. On the other hand, dPCR has a higher precision and has been shown to detect differences of less than 30% in gene expression, distinguish between copy number variations that differ by only 1 copy, and identify alleles that occur at frequencies less than 0.1%.


Applications

Digital PCR has many applications in
basic research Basic research, also called pure research or fundamental research, is a type of scientific research with the aim of improving scientific theories for better understanding and prediction of natural or other phenomena. In contrast, applied resear ...
, clinical diagnostics and environmental testing. Its uses include
pathogen In biology, a pathogen ( el, πάθος, "suffering", "passion" and , "producer of") in the oldest and broadest sense, is any organism or agent that can produce disease. A pathogen may also be referred to as an infectious agent, or simply a germ ...
detection and digestive health analysis;
liquid biopsy A liquid biopsy, also known as fluid biopsy or fluid phase biopsy, is the sampling and analysis of non-solid biological tissue, primarily blood. Like traditional biopsy, this type of technique is mainly used as a diagnostic and monitoring tool for ...
for
cancer Cancer is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. These contrast with benign tumors, which do not spread. Possible signs and symptoms include a lump, abnormal b ...
monitoring, organ
transplant rejection Transplant rejection occurs when Organ transplant, transplanted tissue is rejected by the recipient's immune system, which destroys the transplanted tissue. Transplant rejection can be lessened by determining the molecular similitude between don ...
monitoring and non-invasive
prenatal testing Prenatal testing consists of prenatal screening and prenatal diagnosis, which are aspects of prenatal care that focus on detecting problems with the pregnancy as early as possible. These may be anatomic and physiologic problems with the health of ...
for serious
genetic abnormalities A genetic disorder is a health problem caused by one or more abnormalities in the genome. It can be caused by a mutation in a single gene (monogenic) or multiple genes (polygenic) or by a chromosomal abnormality. Although polygenic disorders ...
;
copy number variation Copy number variation (CNV) is a phenomenon in which sections of the genome are repeated and the number of repeats in the genome varies between individuals. Copy number variation is a type of structural variation: specifically, it is a type of d ...
analysis, single gene expression analysis, rare sequence detection,
gene expression profiling In the field of molecular biology, gene expression profiling is the measurement of the activity (the expression) of thousands of genes at once, to create a global picture of cellular function. These profiles can, for example, distinguish between c ...
and
single-cell analysis In the field of cellular biology, single-cell analysis is the study of genomics, transcriptomics, proteomics, metabolomics and cell–cell interactions at the single cell level. The concept of single-cell analysis originated in the ...
; the 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 Massive parallel sequencing or massively parallel sequencing is any of several high-throughput approaches to DNA sequencing using the concept of massively parallel processing; it is also called next-generation sequencing (NGS) or second-generation s ...
(NGS) and to validate NGS
libraries A library is a collection of materials, books or media that are accessible for use and not just for display purposes. A library provides physical (hard copies) or digital access (soft copies) materials, and may be a physical location or a vir ...
.


Absolute quantification

dPCR enables the absolute and reproducible quantification of target nucleic acids at single-molecule resolution. Unlike analogue
quantitative PCR A real-time polymerase chain reaction (real-time PCR, or qPCR) is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR (i.e., in real ...
(qPCR), however, absolute quantification with dPCR does not require a
standard curve In analytical chemistry, a calibration curve, also known as a standard curve, is a general method for determining the concentration of a substance in an unknown sample by comparing the unknown to a set of standard samples of known concentration. ...
). 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 A genetically modified organism (GMO) is any organism whose genetic material has been altered using genetic engineering techniques. The exact definition of a genetically modified organism and what constitutes genetic engineering varies, with ...
in foodstuffs, viral load in the blood,
PBMC A peripheral blood mononuclear cell (PBMC) is any peripheral blood cell having a round nucleus. These cells consist of lymphocytes (T cells, B cells, NK cells) and monocytes, whereas erythrocytes and platelets have no nuclei, and granulocytes (neu ...
s, serum samples, chorionic villi tissues, biomarkers of neurodegenerative disease in cerebral spinal fluid, and fecal contamination in drinking water.


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 Copy number variation (CNV) is a phenomenon in which sections of the genome are repeated and the number of repeats in the genome varies between individuals. Copy number variation is a type of structural variation: specifically, it is a type of d ...
” (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 The human genome is a complete set of nucleic acid sequences for humans, encoded as DNA within the 23 chromosome pairs in cell nuclei and in a small DNA molecule found within individual mitochondria. These are usually treated separately as the n ...
. 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 Accuracy and precision are two measures of ''observational error''. ''Accuracy'' is how close a given set of measurements ( observations or readings) are to their ''true value'', while ''precision'' is how close the measurements are to each oth ...
when compared to other methods such as SNP-based microarrays 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. In fact, both at these lower target concentrations and at higher ones where multiple copies of the same target can co-localize to a single partition,
Poisson statistics In probability theory and statistics, the Poisson distribution is a discrete probability distribution that expresses the probability of a given number of events occurring in a fixed interval of time or space if these events occur with a known co ...
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 Receptor tyrosine-protein kinase erbB-2 is a protein that in humans is encoded by the ''ERBB2'' gene. ERBB is abbreviated from erythroblastic oncogene B, a gene originally isolated from the avian genome. The human protein is also frequently refer ...
(ERBB2) and
breast cancer Breast cancer is cancer that develops from breast tissue. Signs of breast cancer may include a lump in the breast, a change in breast shape, dimpling of the skin, milk rejection, fluid coming from the nipple, a newly inverted nipple, or a re ...
progression.


Rare mutation and rare allele detection

Partitioning in digital PCR increases sensitivity and allows for detection of rare events, especially
single nucleotide variants In genetics, a single-nucleotide polymorphism (SNP ; plural SNPs ) is a germline substitution of a single nucleotide at a specific position in the genome. Although certain definitions require the substitution to be present in a sufficiently larg ...
(SNVs), by isolating or greatly diminishing the target
biomarker In biomedical contexts, a biomarker, or biological marker, is a measurable indicator of some biological state or condition. Biomarkers are often measured and evaluated using blood, urine, or soft tissues to examine normal biological processes, ...
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 The wild type (WT) is the phenotype of the typical form of a species as it occurs in nature. Originally, the wild type was conceptualized as a product of the standard "normal" allele at a locus, in contrast to that produced by a non-standard, "m ...
background, which is 2,000 times more sensitive than achievable with conventional qPCR.


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.


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 liquid biopsy, also known as fluid biopsy or fluid phase biopsy, is the sampling and analysis of non-solid biological tissue, primarily blood. Like traditional biopsy, this type of technique is mainly used as a diagnostic and monitoring tool for ...
, 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 Cancer is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. These contrast with benign tumors, which do not spread. Possible signs and symptoms include a lump, abnormal b ...
patients by measuring rare mutations in
circulating tumor DNA Circulating tumor DNA (ctDNA) is tumor-derived fragmented DNA in the bloodstream that is not associated with cells. ctDNA should not be confused with cell-free DNA (cfDNA), a broader term which describes DNA that is freely circulating in the bloo ...
(ctDNA) in a variety of biological fluids from patients including
blood Blood is a body fluid in the circulatory system of humans and other vertebrates that delivers necessary substances such as nutrients and oxygen to the cells, and transports metabolic waste products away from those same cells. Blood in the c ...
,
urine Urine is a liquid by-product of metabolism in humans and in many other animals. Urine flows from the kidneys through the ureters to the urinary bladder. Urination results in urine being excretion, excreted from the body through the urethra. Cel ...
and
cerebrospinal fluid Cerebrospinal fluid (CSF) is a clear, colorless body fluid found within the tissue that surrounds the brain and spinal cord of all vertebrates. CSF is produced by specialised ependymal cells in the choroid plexus of the ventricles of the bra ...
. Early detection of ctDNA (as in molecular
relapse In internal medicine, relapse or recidivism is a recurrence of a past (typically medical) condition. For example, multiple sclerosis and malaria often exhibit peaks of activity and sometimes very long periods of dormancy, followed by relapse or r ...
) may lead to earlier administration of an
immunotherapy Immunotherapy or biological therapy is the treatment of disease by activating or suppressing the immune system. Immunotherapies designed to elicit or amplify an immune response are classified as ''activation immunotherapies,'' while immunotherap ...
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 A biopsy is a medical test commonly performed by a surgeon, interventional radiologist, or an interventional cardiologist. The process involves extraction of sample cells or tissues for examination to determine the presence or extent of a diseas ...
data. 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 Non-small-cell lung cancer (NSCLC) is any type of epithelial lung cancer other than small-cell lung carcinoma (SCLC). NSCLC accounts for about 85% of all lung cancers. As a class, NSCLCs are relatively insensitive to chemotherapy, compared to sm ...
. The application of liquid biopsy tests have also been studied in patients with
breast The breast is one of two prominences located on the upper ventral region of a primate's torso. Both females and males develop breasts from the same embryological tissues. In females, it serves as the mammary gland, which produces and secret ...
,
colorectal The large intestine, also known as the large bowel, is the last part of the gastrointestinal tract and of the digestive system in tetrapods. Water is absorbed here and the remaining waste material is stored in the rectum as feces before being ...
,
gynecologic Gynaecology or gynecology (see spelling differences) is the area of medicine that involves the treatment of women's diseases, especially those of the reproductive organs. It is often paired with the field of obstetrics, forming the combined are ...
, and
bladder The urinary bladder, or simply bladder, is a hollow organ in humans and other vertebrates that stores urine from the kidneys before disposal by urination. In humans the bladder is a distensible organ that sits on the pelvic floor. Urine enters ...
cancers to monitor both the disease load and the tumor’s response to treatment.


Gene expression and RNA quantification

Gene expression Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product that enables it to produce end products, protein or non-coding RNA, and ultimately affect a phenotype, as the final effect. The ...
and
RNA Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. RNA and deoxyribonucleic acid ( DNA) are nucleic acids. Along with lipids, proteins, and carbohydra ...
quantification studies have benefited from the increased precision and absolute quantification of dPCR. RNA quantification can be accomplished via
RT-PCR Reverse transcription polymerase chain reaction (RT-PCR) is a laboratory technique combining reverse transcription of RNA into DNA (in this context called complementary DNA or cDNA) and amplification of specific DNA targets using polymerase cha ...
, wherein RNA is reverse-transcribed into
cDNA In genetics, complementary DNA (cDNA) is DNA synthesized from a single-stranded RNA (e.g., messenger RNA (mRNA) or microRNA (miRNA)) template in a reaction catalyzed by the enzyme reverse transcriptase. cDNA is often used to express a speci ...
in the partitioned reaction itself, and the number of RNA molecules originating from each transcript (or allelic transcript) is quantified via dPCR. 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. dPCR is also more resilient to PCR inhibitors for the quantification of RNA than qPCR. 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. 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 Telomerase reverse transcriptase (abbreviated to TERT, or hTERT in humans) is a catalytic subunit of the enzyme telomerase, which, together with the telomerase RNA component (TERC), comprises the most important unit of the telomerase complex. T ...
, 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 In molecular biology, an amplicon is a piece of DNA or RNA that is the source and/or product of amplification or replication events. It can be formed artificially, using various methods including polymerase chain reactions (PCR) or ligase chai ...
for
NGS NGS may refer to: Places * NGSO (NGS orbit), non-geostationary orbit * Nagasaki Airport (IATA airport code: NGS) in Omura, Nagasaki, Japan Organisations * National Galleries of Scotland, representing the national art collection of Scotland * Na ...
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
aptamer Aptamers are short sequences of artificial DNA, RNA, XNA, or peptide that bind a specific target molecule, or family of target molecules. They exhibit a range of affinities ( KD in the pM to μM range), with little or no off-target binding ...
s 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).


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 An emulsion is a mixture of two or more liquids that are normally immiscible (unmixable or unblendable) owing to liquid-liquid phase separation. Emulsions are part of a more general class of two-phase systems of matter called colloids. Althoug ...
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.


Chip-based digital PCR

Chip-based Digital PCR (dPCR) is also a method of dPCR in which the reaction mix (also when used in qPCR) is divided into ~10,000 to ~45,000 partitions on a chip, then amplified using an endpoint PCR thermocycling machine, and is read using a high-powered camera reader with fluorescence filter (HEX, FAM, Cy5, Cy5.5 and Texas Red) for all partitions on each chip.


History

dPCR rose out of an approach first published in 1988 by
Cetus Corporation Cetus Corporation was one of the first biotechnology companies. It was established in Berkeley, California, in 1971, but conducted most of its operations in nearby Emeryville. Before merging with Chiron Corporation in 1991 (now a part of Novarti ...
when researchers showed that a single copy of the β-globin gene could be detected and amplified by PCR. This was achieved by diluting DNA samples from a normal human cell line with DNA from a mutant line having a homozygous deletion of the β-globin gene, until it was no longer present in the reaction. In 1989, Peter Simmonds, AJ Brown et al. used this concept to quantify a molecule for the first time. Alex Morley and Pamela Sykes formally established the method as a quantitative technique in 1992. In 1999, Bert Vogelstein and Kenneth Kinzler coined the term “digital PCR” and showed that the technique could be used to find rare cancer mutations. However, dPCR was difficult to perform; it was labor intensive, required a lot of training to do properly, and was difficult to do in large quantities. In 2003, Kinzler and Vogelstein continued to refine dPCR and created an improved method that they called
BEAMing BEAMing, which stands for beads, emulsion, amplification, magnetics, is a highly sensitive digital PCR method that combines emulsion PCR and flow cytometry to identify and quantify specific somatic mutations present in DNA. Process BEAMing begin ...
technology, an acronym for “beads, emulsion, amplification and magnetics.” The new protocol used emulsion to compartmentalize amplification reactions in a single tube. This change made it possible for scientists to scale the method to thousands of reactions in a single run. Companies developing commercial dPCR systems have integrated technologies like automated partitioning of samples, digital counting of nucleic acid targets, and increasing droplet count that can help the process be more efficient. In recent years, scientists have developed and commercialized dPCR-based diagnostics for several conditions, including
non-small cell lung cancer Non-small-cell lung cancer (NSCLC) is any type of epithelial lung cancer other than small-cell lung carcinoma (SCLC). NSCLC accounts for about 85% of all lung cancers. As a class, NSCLCs are relatively insensitive to chemotherapy, compared to sm ...
and
Down’s Syndrome Down syndrome or Down's syndrome, also known as trisomy 21, is a genetic disorder caused by the presence of all or part of a third copy of chromosome 21. It is usually associated with physical growth delays, mild to moderate intellectual disa ...
. The first dPCR system for clinical use was CE-marked in 2017 and cleared by the US
Food and Drug Administration The United States Food and Drug Administration (FDA or US FDA) is a List of United States federal agencies, federal agency of the United States Department of Health and Human Services, Department of Health and Human Services. The FDA is respon ...
in 2019, for diagnosing
chronic myeloid leukemia Chronic myelogenous leukemia (CML), also known as chronic myeloid leukemia, is a cancer of the white blood cells. It is a form of leukemia characterized by the increased and unregulated growth of myeloid cells in the bone marrow and the accumulat ...
.


References

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External links


Digital PCR ProtocolHigh Throughput, Nanoliter Quantitative PCRPCR's next frontier
Molecular biology Polymerase chain reaction Laboratory techniques