Digital polymerase chain reaction#Droplet Digital PCR

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{{main|Polymerase chain reaction}}

The polymerase chain reaction method is used to quantify nucleic acids by amplifying a nucleic acid molecule with the enzyme DNA polymerase.{{cite web|url=https://www.ncbi.nlm.nih.gov/probe/docs/techpcr/|title=Polymerase Chain Reaction (PCR)|publisher= National Center for Biotechnology Information, U.S. National Library of Medicine}} 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.{{Cite journal |last1=Higuchi |first1=Russell |last2=Fockler |first2=Carita |last3=Dollinger |first3=Gavin |last4=Watson |first4=Robert |date=September 1993 |title=Kinetic PCR Analysis: Real-time Monitoring of DNA Amplification Reactions |url=https://www.nature.com/articles/nbt0993-1026 |journal=Bio/Technology |language=en |volume=11 |issue=9 |pages=1026–1030 |doi=10.1038/nbt0993-1026 |pmid=7764001 |s2cid=5714001 |issn=1546-1696|url-access=subscription }} 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.

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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.{{cite journal | vauthors = Duewer DL, Kline MC, Romsos EL, Toman B | title = Evaluating droplet digital PCR for the quantification of human genomic DNA: converting copies per nanoliter to nanograms nuclear DNA per microliter | journal = Analytical and Bioanalytical Chemistry | volume = 410 | issue = 12 | pages = 2879–2887 | date = May 2018 | pmid = 29556737 | pmc = 5996397 | doi = 10.1007/s00216-018-0982-1 }}{{cite journal| vauthors = Baker M |title=Digital PCR hits its stride|journal=Nature Methods|volume=9|issue=6|year=2012|pages=541–544|doi=10.1038/nmeth.2027|s2cid=46347563|doi-access=free}} 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.{{cite journal | vauthors = Quan PL, Sauzade M, Brouzes E | title = dPCR: A Technology Review | journal = Sensors | volume = 18 | issue = 4 | pages = 1271 | date = April 2018 | pmid = 29677144 | pmc = 5948698 | doi = 10.3390/s18041271 | doi-access = free | bibcode = 2018Senso..18.1271Q}} The PCR solution is partitioned into smaller units, each with the necessary components for amplification. The partitioned units are then subjected to thermocycling so that each unit may independently undergo PCR amplification. 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, 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.{{cite web | vauthors = Prediger E |title=Digital PCR (dPCR)—What is it and why use it? |url=http://www.idtdna.com/pages/decoded/decoded-articles/core-concepts/decoded/2013/10/21/digital-pcr-(dpcr)-what-is-it-and-why-use-it- |publisher=Integrated DNA Technologies}} 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.{{cite journal | vauthors = Butler DM, Pacold ME, Jordan PS, Richman DD, Smith DM | title = The efficiency of single genome amplification and sequencing is improved by quantitation and use of a bioinformatics tool | journal = Journal of Virological Methods | volume = 162 | issue = 1–2 | pages = 280–283 | date = December 2009 | pmid = 19698751 | pmc = 2761514 | doi = 10.1016/j.jviromet.2009.08.002 }} 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.{{Citation needed|date=November 2022}}

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.

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.

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.{{Citation needed|date=February 2017}} 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. 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.{{cite journal | vauthors = Svec D, Tichopad A, Novosadova V, Pfaffl MW, Kubista M | title = How good is a PCR efficiency estimate: Recommendations for precise and robust qPCR efficiency assessments | journal = Biomolecular Detection and Quantification | volume = 3 | pages = 9–16 | date = March 2015 | pmid = 27077029 | pmc = 4822216 | doi = 10.1016/j.bdq.2015.01.005 }}

Real-time Digital PCR (rdPCR) combines the methodologies of digital PCR (dPCR) and quantitative PCR (qPCR), integrating the precision of dPCR with the real-time analysis capabilities of qPCR. This integration aims to provide enhanced sensitivity, specificity, and the ability for absolute quantification of nucleic acid sequences, contributing to the quantification of genetic material in scientific and clinical research.{{Cite journal |last1=Pavšič |first1=Jernej |last2=Žel |first2=Jana |last3=Milavec |first3=Mojca |date=2016-01-01 |title=Assessment of the real-time PCR and different digital PCR platforms for DNA quantification |url=https://doi.org/10.1007/s00216-015-9107-2 |journal=Analytical and Bioanalytical Chemistry |language=en |volume=408 |issue=1 |pages=107–121 |doi=10.1007/s00216-015-9107-2 |issn=1618-2650 |pmc=4706846 |pmid=26521179}}{{Cite journal |last1=Xu |first1=Jiachen |last2=Duong |first2=Kyra |last3=Yang |first3=Zhenlin |last4=Kaji |first4=Kavanaugh |last5=Ou |first5=Jiajia |last6=Head |first6=Steven R. |last7=Crynen |first7=Gogce |last8=Ordoukhanian |first8=Phillip |last9=Hanna |first9=Lauren |last10=Hanna |first10=Ava |last11=Wang |first11=Yan |last12=Wang |first12=Zhijie |last13=Wang |first13=Jie |date=December 2021 |title=Real-time digital polymerase chain reaction (PCR) as a novel technology improves limit of detection for rare allele assays |journal=Translational Lung Cancer Research |language=en |volume=10 |issue=12 |pages=4336–4352 |doi=10.21037/tlcr-21-728 |doi-access=free |pmid=35070745 |pmc=8743530 |issn=2226-4477}}

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%.{{cite journal | vauthors = Salipante SJ, Jerome KR | title = Digital PCR-An Emerging Technology with Broad Applications in Microbiology | journal = Clinical Chemistry | volume = 66 | issue = 1 | pages = 117–123 | date = January 2020 | pmid = 31704712 | doi = 10.1373/clinchem.2019.304048 | doi-access = free }}

Digital PCR has many applications in basic research, clinical diagnostics and environmental testing. Its uses include pathogen detection and digestive health analysis;{{cite journal | vauthors = Witte AK, Fister S, Mester P, Schoder D, Rossmanith P | title = Evaluation of the performance of quantitative detection of the Listeria monocytogenes prfA locus with droplet digital PCR | journal = Analytical and Bioanalytical Chemistry | volume = 408 | issue = 27 | pages = 7583–7593 | date = November 2016 | pmid = 27558101 | pmc = 5061835 | doi = 10.1007/s00216-016-9861-9 }}{{cite journal | vauthors = Stauber J, Shaikh N, Ordiz MI, Tarr PI, Manary MJ | title = Droplet digital PCR quantifies host inflammatory transcripts in feces reliably and reproducibly | journal = Cellular Immunology | volume = 303 | pages = 43–49 | date = May 2016 | pmid = 27063479 | pmc = 4863679 | doi = 10.1016/j.cellimm.2016.03.007 | author-link = Jenny Stauber }} liquid biopsy for cancer monitoring, organ transplant rejection monitoring and non-invasive prenatal testing for serious genetic abnormalities;{{cite news | vauthors = Skibo S |date=23 Feb 2018 |title= Has Tumor Profiling Caught Up to Cancer?|url=https://thepathologist.com/diagnostics/has-tumor-profiling-caught-up-to-cancer |access-date=23 July 2019}}{{cite news | vauthors = Hirsch F |date=27 July 2018 |title= Guidelines highlight 'best practices' for liquid biopsy during treatment of non-small cell lung cancer|url=https://www.healio.com/hematology-oncology/lung-cancer/news/in-the-journals/%7B7a38fda7-6a5d-4e0b-a78b-60f6a64d6bdc%7D/guidelines-highlight-best-practices-for-liquid-biopsy-during-treatment-of-non-small-cell-lung-cancer |access-date=23 July 2019}}{{cite news | vauthors = Johnson M |date=12 Jan 2018 |title= Bio-Rad Continues to Advance Digital PCR Tech, Liquid Biopsy Tests Into Commercial Clinical Market|url=https://www.genomeweb.com/molecular-diagnostics/bio-rad-continues-advance-digital-pcr-tech-liquid-biopsy-tests-commercial#.XTcmipNKh-V |access-date=23 July 2019}}{{cite journal | vauthors = Oxnard GR, Paweletz CP, Kuang Y, Mach SL, O'Connell A, Messineo MM, Luke JJ, Butaney M, Kirschmeier P, Jackman DM, Jänne PA | display-authors = 6 | title = Noninvasive detection of response and resistance in EGFR-mutant lung cancer using quantitative next-generation genotyping of cell-free plasma DNA | journal = Clinical Cancer Research | volume = 20 | issue = 6 | pages = 1698–1705 | date = March 2014 | pmid = 24429876 | pmc = 3959249 | doi = 10.1158/1078-0432.CCR-13-2482 }}{{cite journal | vauthors = Schütz E, Fischer A, Beck J, Harden M, Koch M, Wuensch T, Stockmann M, Nashan B, Kollmar O, Matthaei J, Kanzow P, Walson PD, Brockmöller J, Oellerich M | display-authors = 6 | title = Graft-derived cell-free DNA, a noninvasive early rejection and graft damage marker in liver transplantation: A prospective, observational, multicenter cohort study | journal = PLOS Medicine | volume = 14 | issue = 4 | pages = e1002286 | date = April 2017 | pmid = 28441386 | pmc = 5404754 | doi = 10.1371/journal.pmed.1002286 | doi-access = free }}{{cite journal| vauthors = Lee SY, Hwang SY |title=Application of digital polymerase chain reaction technology for noninvasive prenatal test|journal= Journal of Genetic Medicine|volume=12|issue=2|pages=72–78|year=2015 |doi=10.5734/JGM.2015.12.2.72 |issn=2383-8442|doi-access=free}}{{cite journal | vauthors = Gu W, Koh W, Blumenfeld YJ, El-Sayed YY, Hudgins L, Hintz SR, Quake SR | title = Noninvasive prenatal diagnosis in a fetus at risk for methylmalonic acidemia | journal = Genetics in Medicine | volume = 16 | issue = 7 | pages = 564–567 | date = July 2014 | pmid = 24406457 | pmc = 4079742 | doi = 10.1038/gim.2013.194 }}{{cite journal | vauthors = Strain MC, Lada SM, Luong T, Rought SE, Gianella S, Terry VH, Spina CA, Woelk CH, Richman DD | display-authors = 6 | title = Highly precise measurement of HIV DNA by droplet digital PCR | journal = PLOS ONE | volume = 8 | issue = 4 | pages = e55943 | year = 2013 | pmid = 23573183 | pmc = 3616050 | doi = 10.1371/journal.pone.0055943 | doi-access = free | bibcode = 2013PLoSO...855943S }} copy number variation analysis,{{cite book | vauthors = Bell AD, Usher CL, McCarroll SA | title = Digital PCR | chapter = Analyzing Copy Number Variation with Droplet Digital PCR | series = Methods in Molecular Biology | location = Clifton, N.J. | volume = 1768 | pages = 143–160 | date = 2018 | pmid = 29717442 | doi = 10.1007/978-1-4939-7778-9_9 |isbn=978-1-4939-7776-5 }}{{cite journal | vauthors = Shoda K, Ichikawa D, Fujita Y, Masuda K, Hiramoto H, Hamada J, Arita T, Konishi H, Komatsu S, Shiozaki A, Kakihara N, Okamoto K, Taniguchi H, Imoto I, Otsuji E | display-authors = 6 | title = Monitoring the HER2 copy number status in circulating tumor DNA by droplet digital PCR in patients with gastric cancer | journal = Gastric Cancer | volume = 20 | issue = 1 | pages = 126–135 | date = January 2017 | pmid = 26874951 | doi = 10.1007/s10120-016-0599-z | doi-access = free }}{{cite journal | vauthors = Gevensleben H, Garcia-Murillas I, Graeser MK, Schiavon G, Osin P, Parton M, Smith IE, Ashworth A, Turner NC | display-authors = 6 | title = Noninvasive detection of HER2 amplification with plasma DNA digital PCR | journal = Clinical Cancer Research | volume = 19 | issue = 12 | pages = 3276–3284 | date = June 2013 | pmid = 23637122 | pmc = 6485473 | doi = 10.1158/1078-0432.CCR-12-3768 }} single gene expression analysis,{{cite journal | vauthors = Mazzoni E, Frontini F, Rotondo JC, Zanotta N, Fioravanti A, Minelli F, Torreggiani E, Campisciano G, Marcuzzi A, Guerra G, Tommasini A, Touzé A, Martini F, Tognon M, Comar M | display-authors = 6 | title = Antibodies reacting to mimotopes of Simian virus 40 large T antigen, the viral oncoprotein, in sera from children | journal = Journal of Cellular Physiology | volume = 234 | issue = 4 | pages = 3170–3179 | date = April 2019 | pmid = 30362540 | doi = 10.1002/jcp.27490 | hdl = 11392/2397717 | s2cid = 53106591 | hdl-access = free }} rare sequence detection,{{cite journal | vauthors = Uchiyama Y, Nakashima M, Watanabe S, Miyajima M, Taguri M, Miyatake S, Miyake N, Saitsu H, Mishima H, Kinoshita A, Arai H, Yoshiura K, Matsumoto N | display-authors = 6 | title = Ultra-sensitive droplet digital PCR for detecting a low-prevalence somatic GNAQ mutation in Sturge-Weber syndrome | journal = Scientific Reports | volume = 6 | issue = 1 | pages = 22985 | date = March 2016 | pmid = 26957145 | pmc = 4783707 | doi = 10.1038/srep22985 | bibcode = 2016NatSR...622985U }}{{cite news | vauthors = Marusina K |date=1 Oct 2017 |title= Positioning Digital PCR for Sharper Genomic Views |url=https://www.genengnews.com/magazine/positioning-digital-pcr-for-sharper-genomic-views/ |access-date=23 July 2019}} gene expression profiling and single-cell analysis;{{cite book | vauthors = Kamitaki N, Usher CL, McCarroll SA | title = Digital PCR | chapter = Using Droplet Digital PCR to Analyze Allele-Specific RNA Expression | series = Methods in Molecular Biology | volume = 1768 | pages = 401–422 | year = 2018 | pmid = 29717456 | doi = 10.1007/978-1-4939-7778-9_23 | isbn = 978-1-4939-7776-5 }}{{cite journal | vauthors = Millier MJ, Stamp LK, Hessian PA | title = Digital-PCR for gene expression: impact from inherent tissue RNA degradation | journal = Scientific Reports | volume = 7 | issue = 1 | pages = 17235 | date = December 2017 | pmid = 29222437 | pmc = 5722939 | doi = 10.1038/s41598-017-17619-0 | bibcode = 2017NatSR...717235M }}{{cite news |date=12 Apr 2018 |title= Highly Sensitive Detection of Hepatitis B Using ddPCR |url=https://www.genengnews.com/topics/omics/highly-sensitive-detection-of-hepatitis-b-using-ddpcr/ |access-date=23 July 2019}}{{cite journal| vauthors = Jang M, Jeong SW, Bae NH, Song Y, Lee TJ, Lee MK, Lee SJ, Lee KG |title=Droplet-based digital PCR system for detection of single-cell level of foodborne pathogens|journal=BioChip Journal|volume=11|issue=4|pages=329–337|year=2017|issn=2092-7843|doi=10.1007/s13206-017-1410-x|s2cid=89829687}}{{cite journal | vauthors = Igarashi Y, Uchiyama T, Minegishi T, Takahashi S, Watanabe N, Kawai T, Yamada M, Ariga T, Onodera M | display-authors = 6 | title = Single Cell-Based Vector Tracing in Patients with ADA-SCID Treated with Stem Cell Gene Therapy | journal = Molecular Therapy. Methods & Clinical Development | volume = 6 | pages = 8–16 | date = September 2017 | pmid = 28626778 | pmc = 5466583 | doi = 10.1016/j.omtm.2017.05.005 }}{{cite journal | vauthors = Albayrak C, Jordi CA, Zechner C, Lin J, Bichsel CA, Khammash M, Tay S | title = Digital Quantification of Proteins and mRNA in Single Mammalian Cells | journal = Molecular Cell | volume = 61 | issue = 6 | pages = 914–924 | date = March 2016 | pmid = 26990994 | doi = 10.1016/j.molcel.2016.02.030 | doi-access = free }} the detection of DNA contaminants in bioprocessing,{{cite journal | vauthors = Hussain M, Fantuzzo R, Mercorelli S, Cullen C | title = A direct droplet digital PCR method for quantification of residual DNA in protein drugs produced in yeast cells | journal = Journal of Pharmaceutical and Biomedical Analysis | volume = 123 | pages = 128–131 | date = May 2016 | pmid = 26896631 | doi = 10.1016/j.jpba.2016.01.050 }} the validation of gene edits and detection of specific methylation changes in DNA as biomarkers of cancer,{{cite journal | vauthors = Miyaoka Y, Chan AH, Judge LM, Yoo J, Huang M, Nguyen TD, Lizarraga PP, So PL, Conklin BR | display-authors = 6 | title = Isolation of single-base genome-edited human iPS cells without antibiotic selection | journal = Nature Methods | volume = 11 | issue = 3 | pages = 291–293 | date = March 2014 | pmid = 24509632 | pmc = 4063274 | doi = 10.1038/nmeth.2840 }}{{cite journal | vauthors = Nelson CE, Hakim CH, Ousterout DG, Thakore PI, Moreb EA, Castellanos Rivera RM, Madhavan S, Pan X, Ran FA, Yan WX, Asokan A, Zhang F, Duan D, Gersbach CA | display-authors = 6 | title = In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy | journal = Science | volume = 351 | issue = 6271 | pages = 403–407 | date = January 2016 | pmid = 26721684 | pmc = 4883596 | doi = 10.1126/science.aad5143 | bibcode = 2016Sci...351..403N }}{{cite journal | vauthors = Miyaoka Y, Berman JR, Cooper SB, Mayerl SJ, Chan AH, Zhang B, Karlin-Neumann GA, Conklin BR | display-authors = 6 | title = Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell type on genome-editing | journal = Scientific Reports | volume = 6 | pages = 23549 | date = March 2016 | pmid = 27030102 | pmc = 4814844 | doi = 10.1038/srep23549 | bibcode = 2016NatSR...623549M }} as well as plasmid copy number determination in bacterial populations.{{Cite journal |last1=Nicoloff |first1=Hervé |last2=Hjort |first2=Karin |last3=Andersson |first3=Dan I. |last4=Wang |first4=Helen |date=2024-05-10 |title=Three concurrent mechanisms generate gene copy number variation and transient antibiotic heteroresistance |journal=Nature Communications |language=en |volume=15 |issue=1 |pages=3981 |doi=10.1038/s41467-024-48233-0 |issn=2041-1723 |pmc=11087502 |pmid=38730266|bibcode=2024NatCo..15.3981N }} dPCR is also frequently used as an orthogonal method to confirm rare mutations detected through next-generation sequencing (NGS) and to validate NGS libraries.{{cite journal | vauthors = Guttery DS, Page K, Hills A, Woodley L, Marchese SD, Rghebi B, Hastings RK, Luo J, Pringle JH, Stebbing J, Coombes RC, Ali S, Shaw JA | display-authors = 6 | title = Noninvasive detection of activating estrogen receptor 1 (ESR1) mutations in estrogen receptor-positive metastatic breast cancer | journal = Clinical Chemistry | volume = 61 | issue = 7 | pages = 974–982 | date = July 2015 | pmid = 25979954 | doi = 10.1373/clinchem.2015.238717 | doi-access = free }}{{cite journal | vauthors = Robin JD, Ludlow AT, LaRanger R, Wright WE, Shay JW | title = Comparison of DNA Quantification Methods for Next Generation Sequencing | journal = Scientific Reports | volume = 6 | issue = 1 | pages = 24067 | date = April 2016 | pmid = 27048884 | pmc = 4822169 | doi = 10.1038/srep24067 | bibcode = 2016NatSR...624067R }}{{cite journal | vauthors = Aigrain L, Gu Y, Quail MA | title = Quantitation of next generation sequencing library preparation protocol efficiencies using droplet digital PCR assays - a systematic comparison of DNA library preparation kits for Illumina sequencing | journal = BMC Genomics | volume = 17 | issue = 1 | pages = 458 | date = June 2016 | pmid = 27297323 | pmc = 4906846 | doi = 10.1186/s12864-016-2757-4 | doi-access = free }}

dPCR enables the absolute and reproducible quantification of target nucleic acids at single-molecule resolution.{{cite journal | vauthors = Brunetto GS, Massoud R, Leibovitch EC, Caruso B, Johnson K, Ohayon J, Fenton K, Cortese I, Jacobson S | display-authors = 6 | title = Digital droplet PCR (ddPCR) for the precise quantification of human T-lymphotropic virus 1 proviral loads in peripheral blood and cerebrospinal fluid of HAM/TSP patients and identification of viral mutations | journal = Journal of Neurovirology | volume = 20 | issue = 4 | pages = 341–351 | date = August 2014 | pmid = 24781526 | pmc = 4085507 | doi = 10.1007/s13365-014-0249-3 }}{{cite journal | vauthors = Vogelstein B, Kinzler KW | title = Digital PCR | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 96 | issue = 16 | pages = 9236–9241 | date = August 1999 | pmid = 10430926 | pmc = 17763 | doi = 10.1073/pnas.96.16.9236 | doi-access = free | bibcode = 1999PNAS...96.9236V }} Unlike analogue quantitative PCR (qPCR), however, absolute quantification with dPCR does not require a standard curve. dPCR also has a greater tolerance for inhibitor substances and PCR assays that amplify inefficiently as compared to qPCR.{{cite journal | vauthors = Rački N, Dreo T, Gutierrez-Aguirre I, Blejec A, Ravnikar M | title = Reverse transcriptase droplet digital PCR shows high resilience to PCR inhibitors from plant, soil and water samples | journal = Plant Methods | volume = 10 | issue = 1 | pages = 42 | year = 2014 | pmid = 25628753 | pmc = 4307183 | doi = 10.1186/s13007-014-0042-6 | doi-access = free }}{{cite journal | vauthors = Dingle TC, Sedlak RH, Cook L, Jerome KR | title = Tolerance of droplet-digital PCR vs real-time quantitative PCR to inhibitory substances | journal = Clinical Chemistry | volume = 59 | issue = 11 | pages = 1670–1672 | date = November 2013 | pmid = 24003063 | pmc = 4247175 | doi = 10.1373/clinchem.2013.211045 }}

dPCR can quantify, for example, the presence of specific sequences from contaminating genetically modified organisms in foodstuffs,{{cite book | vauthors = Dobnik D, Spilsberg B, Bogožalec Košir A, Štebih D, Morisset D, Holst-Jensen A, Žel J | title = Digital PCR | chapter = Multiplex Droplet Digital PCR Protocols for Quantification of GM Maize Events | series = Methods in Molecular Biology | volume = 1768 | pages = 69–98 | year = 2018 | pmid = 29717438 | doi = 10.1007/978-1-4939-7778-9_5 | isbn = 978-1-4939-7776-5 }} viral load in the blood,{{cite book | vauthors = Vellucci A, Leibovitch EC, Jacobson S | title = Digital PCR | chapter = Using Droplet Digital PCR to Detect Coinfection of Human Herpesviruses 6A and 6B (HHV-6A and HHV-6B) in Clinical Samples | series = Methods in Molecular Biology | volume = 1768 | pages = 99–109 | year = 2018 | pmid = 29717439 | doi = 10.1007/978-1-4939-7778-9_6 | isbn = 978-1-4939-7776-5 }} PBMCs,{{cite journal | vauthors = Tagliapietra A, Rotondo JC, Bononi I, Mazzoni E, Magagnoli F, Gonzalez LO, Contini C, Vesce F, Tognon M, Martini F | display-authors = 6 | title = Droplet-digital PCR assay to detect Merkel cell polyomavirus sequences in chorionic villi from spontaneous abortion affected females | journal = Journal of Cellular Physiology | volume = 235 | issue = 3 | pages = 1888–1894 | date = March 2020 | pmid = 31549405 | doi = 10.1002/jcp.29213 | doi-access = free | hdl = 11392/2409453 | hdl-access = free }}{{cite journal | vauthors = Tagliapietra A, Rotondo JC, Bononi I, Mazzoni E, Magagnoli F, Maritati M, Contini C, Vesce F, Tognon M, Martini F | display-authors = 6 | title = Footprints of BK and JC polyomaviruses in specimens from females affected by spontaneous abortion | journal = Human Reproduction | volume = 34 | issue = 3 | pages = 433–440 | date = March 2019 | pmid = 30590693 | doi = 10.1002/jcp.27490 | hdl = 11392/2397717 | s2cid = 53106591 | hdl-access = free }} serum samples,{{cite journal | vauthors = Mazzoni E, Rotondo JC, Marracino L, Selvatici R, Bononi I, Torreggiani E, Touzé A, Martini F, Tognon MG | display-authors = 6 | title = Detection of Merkel Cell Polyomavirus DNA in Serum Samples of Healthy Blood Donors | journal = Frontiers in Oncology | volume = 7 | pages = 294 | date = 2017 | pmid = 29238698 | pmc = 5712532 | doi = 10.3389/fonc.2017.00294 | doi-access = free }} chorionic villi tissues, biomarkers of neurodegenerative disease in cerebral spinal fluid,{{cite book | vauthors = Podlesniy P, Trullas R | title = Digital PCR | chapter = Biomarkers in Cerebrospinal Fluid: Analysis of Cell-Free Circulating Mitochondrial DNA by Digital PCR | series = Methods in Molecular Biology | volume = 1768 | pages = 111–126 | year = 2018 | pmid = 29717440 | doi = 10.1007/978-1-4939-7778-9_7 | isbn = 978-1-4939-7776-5 }} and fecal contamination in drinking water.{{cite book | vauthors = Cao Y, Raith MR, Griffith JF | title = Digital PCR | chapter = Testing of General and Human-Associated Fecal Contamination in Waters | series = Methods in Molecular Biology | volume = 1768 | pages = 127–140 | year = 2018 | pmid = 29717441 | doi = 10.1007/978-1-4939-7778-9_8 | isbn = 978-1-4939-7776-5 }}

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.{{cite journal | vauthors = Li W, Lee A, Gregersen PK | title = Copy-number-variation and copy-number-alteration region detection by cumulative plots | journal = BMC Bioinformatics | volume = 10 | issue = S1 | pages = S67 | date = January 2009 | pmid = 19208171 | pmc = 2648736 | doi = 10.1186/1471-2105-10-S1-S67 | arxiv = 0909.3129 | bibcode = 2009arXiv0909.3129L | doi-access = free }} 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.{{cite journal | vauthors = Koren A, Handsaker RE, Kamitaki N, Karlić R, Ghosh S, Polak P, Eggan K, McCarroll SA | display-authors = 6 | title = Genetic variation in human DNA replication timing | journal = Cell | volume = 159 | issue = 5 | pages = 1015–1026 | date = November 2014 | pmid = 25416942 | pmc = 4359889 | doi = 10.1016/j.cell.2014.10.025 }}{{cite news | vauthors = Sanders S |date=16 Jul 2008 |title= CNVs vs SNPs: Understanding Human Structural Variation in Disease|url=https://www.sciencemag.org/custom-publishing/webinars/cnvs-vs-snps-understanding-human-structural-variation-disease |access-date=24 July 2019}}{{cite journal | vauthors = Marshall CR, Howrigan DP, Merico D, Thiruvahindrapuram B, Wu W, Greer DS, Antaki D, Shetty A, Holmans PA, Pinto D, Gujral M, Brandler WM, Malhotra D, Wang Z, Fajarado KV, Maile MS, Ripke S, Agartz I, Albus M, Alexander M, Amin F, Atkins J, Bacanu SA, Belliveau RA, Bergen SE, Bertalan M, Bevilacqua E, Bigdeli TB, Black DW, Bruggeman R, Buccola NG, Buckner RL, Bulik-Sullivan B, Byerley W, Cahn W, Cai G, Cairns MJ, Campion D, Cantor RM, Carr VJ, Carrera N, Catts SV, Chambert KD, Cheng W, Cloninger CR, Cohen D, Cormican P, Craddock N, Crespo-Facorro B, Crowley JJ, Curtis D, Davidson M, Davis KL, Degenhardt F, Del Favero J, DeLisi LE, Dikeos D, Dinan T, Djurovic S, Donohoe G, Drapeau E, Duan J, Dudbridge F, Eichhammer P, Eriksson J, Escott-Price V, Essioux L, Fanous AH, Farh KH, Farrell MS, Frank J, Franke L, Freedman R, Freimer NB, Friedman JI, Forstner AJ, Fromer M, Genovese G, Georgieva L, Gershon ES, Giegling I, Giusti-Rodríguez P, Godard S, Goldstein JI, Gratten J, de Haan L, Hamshere ML, Hansen M, Hansen T, Haroutunian V, Hartmann AM, Henskens FA, Herms S, Hirschhorn JN, Hoffmann P, Hofman A, Huang H, Ikeda M, Joa I, Kähler AK, Kahn RS, Kalaydjieva L, Karjalainen J, Kavanagh D, Keller MC, Kelly BJ, Kennedy JL, Kim Y, Knowles JA, Konte B, Laurent C, Lee P, Lee SH, Legge SE, Lerer B, Levy DL, Liang KY, Lieberman J, Lönnqvist J, Loughland CM, Magnusson PK, Maher BS, Maier W, Mallet J, Mattheisen M, Mattingsdal M, McCarley RW, McDonald C, McIntosh AM, Meier S, Meijer CJ, Melle I, Mesholam-Gately RI, Metspalu A, Michie PT, Milani L, Milanova V, Mokrab Y, Morris DW, Müller-Myhsok B, Murphy KC, Murray RM, Myin-Germeys I, Nenadic I, Nertney DA, Nestadt G, Nicodemus KK, Nisenbaum L, Nordin A, O'Callaghan E, O'Dushlaine C, Oh SY, Olincy A, Olsen L, O'Neill FA, Van Os J, Pantelis C, Papadimitriou GN, Parkhomenko E, Pato MT, Paunio T, Perkins DO, Pers TH, Pietiläinen O, Pimm J, Pocklington AJ, Powell J, Price A, Pulver AE, Purcell SM, Quested D, Rasmussen HB, Reichenberg A, Reimers MA, Richards AL, Roffman JL, Roussos P, Ruderfer DM, Salomaa V, Sanders AR, Savitz A, Schall U, Schulze TG, Schwab SG, Scolnick EM, Scott RJ, Seidman LJ, Shi J, Silverman JM, Smoller JW, Söderman E, Spencer CC, Stahl EA, Strengman E, Strohmaier J, Stroup TS, Suvisaari J, Svrakic DM, Szatkiewicz JP, Thirumalai S, Tooney PA, Veijola J, Visscher PM, Waddington J, Walsh D, Webb BT, Weiser M, Wildenauer DB, Williams NM, Williams S, Witt SH, Wolen AR, Wormley BK, Wray NR, Wu JQ, Zai CC, Adolfsson R, Andreassen OA, Blackwood DH, Bramon E, Buxbaum JD, Cichon S, Collier DA, Corvin A, Daly MJ, Darvasi A, Domenici E, Esko T, Gejman PV, Gill M, Gurling H, Hultman CM, Iwata N, Jablensky AV, Jönsson EG, Kendler KS, Kirov G, Knight J, Levinson DF, Li QS, McCarroll SA, McQuillin A, Moran JL, Mowry BJ, Nöthen MM, Ophoff RA, Owen MJ, Palotie A, Pato CN, Petryshen TL, Posthuma D, Rietschel M, Riley BP, Rujescu D, Sklar P, St Clair D, Walters JT, Werge T, Sullivan PF, O'Donovan MC, Scherer SW, Neale BM, Sebat J | display-authors = 6 | title = Contribution of copy number variants to schizophrenia from a genome-wide study of 41,321 subjects | journal = Nature Genetics | volume = 49 | issue = 1 | pages = 27–35 | date = January 2017 | pmid = 27869829 | pmc = 5737772 | doi = 10.1038/ng.3725 }} They have been associated with cancers;{{cite journal | vauthors = Shlien A, Malkin D | title = Copy number variations and cancer | journal = Genome Medicine | volume = 1 | issue = 6 | pages = 62 | date = June 2009 | pmid = 19566914 | pmc = 2703871 | doi = 10.1186/gm62 | doi-access = free }}{{cite journal | vauthors = Lauer S, Gresham D | title = An evolving view of copy number variants | journal = Current Genetics | volume = 65 | issue = 6 | pages = 1287–1295 | date = December 2019 | pmid = 31076843 | doi = 10.1007/s00294-019-00980-0 | s2cid = 149444714 }}{{cite news |date=5 Sep 2018 |title= Copy Number Alteration Found to Be Associated with Cancer Mortality |url=https://www.genengnews.com/news/copy-number-alteration-found-to-be-associated-with-cancer-mortality/|access-date=24 July 2019}} neurological,{{cite journal | vauthors = Gu W, Lupski JR | title = CNV and nervous system diseases--what's new? | journal = Cytogenetic and Genome Research | volume = 123 | issue = 1–4 | pages = 54–64 | year = 2008 | pmid = 19287139 | pmc = 2920183 | doi = 10.1159/000184692 }} psychiatric,{{cite journal | vauthors = Thapar A, Cooper M | title = Copy number variation: what is it and what has it told us about child psychiatric disorders? | journal = Journal of the American Academy of Child and Adolescent Psychiatry | volume = 52 | issue = 8 | pages = 772–774 | date = August 2013 | pmid = 23880486 | pmc = 3919207 | doi = 10.1016/j.jaac.2013.05.013 }}{{cite journal | vauthors = Sekar A, Bialas AR, de Rivera H, Davis A, Hammond TR, Kamitaki N, Tooley K, Presumey J, Baum M, Van Doren V, Genovese G, Rose SA, Handsaker RE, Daly MJ, Carroll MC, Stevens B, McCarroll SA | display-authors = 6 | title = Schizophrenia risk from complex variation of complement component 4 | journal = Nature | volume = 530 | issue = 7589 | pages = 177–183 | date = February 2016 | pmid = 26814963 | pmc = 4752392 | doi = 10.1038/nature16549 | bibcode = 2016Natur.530..177. }} and autoimmune diseases;{{cite journal | vauthors = Yim SH, Jung SH, Chung B, Chung YJ | title = Clinical implications of copy number variations in autoimmune disorders | journal = The Korean Journal of Internal Medicine | volume = 30 | issue = 3 | pages = 294–304 | date = May 2015 | pmid = 25995659 | pmc = 4438283 | doi = 10.3904/kjim.2015.30.3.294 }} and adverse drug reactions.{{cite journal | vauthors = He Y, Hoskins JM, McLeod HL | title = Copy number variants in pharmacogenetic genes | journal = Trends in Molecular Medicine | volume = 17 | issue = 5 | pages = 244–251 | date = May 2011 | pmid = 21388883 | pmc = 3092840 | doi = 10.1016/j.molmed.2011.01.007 }} 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.{{cite journal | vauthors = Gonzalez E, Kulkarni H, Bolivar H, Mangano A, Sanchez R, Catano G, Nibbs RJ, Freedman BI, Quinones MP, Bamshad MJ, Murthy KK, Rovin BH, Bradley W, Clark RA, Anderson SA, O'connell RJ, Agan BK, Ahuja SS, Bologna R, Sen L, Dolan MJ, Ahuja SK | display-authors = 6 | title = The influence of CCL3L1 gene-containing segmental duplications on HIV-1/AIDS susceptibility | journal = Science | volume = 307 | issue = 5714 | pages = 1434–1440 | date = March 2005 | pmid = 15637236 | doi = 10.1126/science.1101160 | s2cid = 8815153 | bibcode = 2005Sci...307.1434G }}{{cite journal | vauthors = Liu S, Yao L, Ding D, Zhu H | title = CCL3L1 copy number variation and susceptibility to HIV-1 infection: a meta-analysis | journal = PLOS ONE | volume = 5 | issue = 12 | pages = e15778 | date = December 2010 | pmid = 21209899 | pmc = 3012711 | doi = 10.1371/journal.pone.0015778 | doi-access = free | bibcode = 2010PLoSO...515778L }}

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{{cite journal | vauthors = Dube S, Qin J, Ramakrishnan R | title = Mathematical analysis of copy number variation in a DNA sample using digital PCR on a nanofluidic device | journal = PLOS ONE | volume = 3 | issue = 8 | pages = e2876 | date = August 2008 | pmid = 18682853 | pmc = 2483940 | doi = 10.1371/journal.pone.0002876 | doi-access = free | bibcode = 2008PLoSO...3.2876D }} or qPCR.{{cite journal | vauthors = Hughesman CB, Lu XJ, Liu KY, Zhu Y, Towle RM, Haynes C, Poh CF | title = Detection of clinically relevant copy number alterations in oral cancer progression using multiplexed droplet digital PCR | journal = Scientific Reports | volume = 7 | issue = 1 | pages = 11855 | date = September 2017 | pmid = 28928368 | pmc = 5605662 | doi = 10.1038/s41598-017-11201-4 | bibcode = 2017NatSR...711855H }}{{cite journal | vauthors = Usher CL, Handsaker RE, Esko T, Tuke MA, Weedon MN, Hastie AR, Cao H, Moon JE, Kashin S, Fuchsberger C, Metspalu A, Pato CN, Pato MT, McCarthy MI, Boehnke M, Altshuler DM, Frayling TM, Hirschhorn JN, McCarroll SA | display-authors = 6 | title = Structural forms of the human amylase locus and their relationships to SNPs, haplotypes and obesity | journal = Nature Genetics | volume = 47 | issue = 8 | pages = 921–925 | date = August 2015 | pmid = 26098870 | pmc = 4712930 | doi = 10.1038/ng.3340 }} qPCR is limited in its ability to precisely quantify gene amplifications in several diseases, including Crohn’s disease, HIV-1 infection, and obesity.{{cite journal | vauthors = Aldhous MC, Abu Bakar S, Prescott NJ, Palla R, Soo K, Mansfield JC, Mathew CG, Satsangi J, Armour JA | display-authors = 6 | title = Measurement methods and accuracy in copy number variation: failure to replicate associations of beta-defensin copy number with Crohn's disease | journal = Human Molecular Genetics | volume = 19 | issue = 24 | pages = 4930–4938 | date = December 2010 | pmid = 20858604 | pmc = 2989891 | doi = 10.1093/hmg/ddq411 }}

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 are used to correct for these multiple occupancies to give a more accurate value for each target’s concentration.{{cite book | vauthors = Pinheiro L, Emslie KR | title = Digital PCR | chapter = Basic Concepts and Validation of Digital PCR Measurements | series = Methods in Molecular Biology | volume = 1768 | pages = 11–24 | date = 2018 | pmid = 29717435 | doi = 10.1007/978-1-4939-7778-9_2 | isbn = 978-1-4939-7776-5 }}

Digital PCR has been used to uncover both germline and somatic variation in gene copy number between humans{{cite journal | vauthors = Handsaker RE, Van Doren V, Berman JR, Genovese G, Kashin S, Boettger LM, McCarroll SA | title = Large multiallelic copy number variations in humans | journal = Nature Genetics | volume = 47 | issue = 3 | pages = 296–303 | date = March 2015 | pmid = 25621458 | pmc = 4405206 | doi = 10.1038/ng.3200 }} and to study the link between amplification of HER2 (ERBB2) and breast cancer progression.{{cite book | vauthors = Garcia-Murillas I, Turner NC | title = Digital PCR | chapter = Assessing HER2 Amplification in Plasma cfDNA | series = Methods in Molecular Biology | volume = 1768 | pages = 161–172 | year = 2018 | pmid = 29717443 | doi = 10.1007/978-1-4939-7778-9_10 | isbn = 978-1-4939-7776-5 }}{{cite journal | vauthors = Christgen M, van Luttikhuizen JL, Raap M, Braubach P, Schmidt L, Jonigk D, Feuerhake F, Lehmann U, Schlegelberger B, Kreipe HH, Steinemann D | display-authors = 6 | title = Precise ERBB2 copy number assessment in breast cancer by means of molecular inversion probe array analysis | journal = Oncotarget | volume = 7 | issue = 50 | pages = 82733–82740 | date = December 2016 | pmid = 27716627 | pmc = 5347728 | doi = 10.18632/oncotarget.12421 }}{{cite journal | vauthors = Borley A, Mercer T, Morgan M, Dutton P, Barrett-Lee P, Brunelli M, Jasani B | title = Impact of HER2 copy number in IHC2+/FISH-amplified breast cancer on outcome of adjuvant trastuzumab treatment in a large UK cancer network | journal = British Journal of Cancer | volume = 110 | issue = 8 | pages = 2139–2143 | date = April 2014 | pmid = 24691421 | pmc = 3992505 | doi = 10.1038/bjc.2014.147 }}

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.{{cite journal | vauthors = Pekin D, Skhiri Y, Baret JC, Le Corre D, Mazutis L, Salem CB, Millot F, El Harrak A, Hutchison JB, Larson JW, Link DR, Laurent-Puig P, Griffiths AD, Taly V | display-authors = 6 | title = Quantitative and sensitive detection of rare mutations using droplet-based microfluidics | journal = Lab on a Chip | volume = 11 | issue = 13 | pages = 2156–2166 | date = July 2011 | pmid = 21594292 | doi = 10.1039/c1lc20128j }} These events can be organized into two classes: rare mutation detection and rare sequence 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.

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.{{cite journal | vauthors = Cao Y, Raith MR, Griffith JF | title = Droplet digital PCR for simultaneous quantification of general and human-associated fecal indicators for water quality assessment | journal = Water Research | volume = 70 | pages = 337–349 | date = March 2015 | pmid = 25543243 | doi = 10.1016/j.watres.2014.12.008 | bibcode = 2015WatRe..70..337C }} dPCR can detect sequences as rare as 1 in every 1,250,000 cells.

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.{{cite news |last=European Society for Medical Oncology |date=17 Nov 2017 |title= Study analyzes mutations in cerebrospinal fluid in lung cancer with brain metastases|url=https://medicalxpress.com/news/2017-11-mutations-cerebrospinal-fluid-lung-cancer.html |access-date=24 July 2019}} 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.{{cite news | vauthors = Petrone J | date = 8 June 2017 |title= Norwegian Team Plans to Debut Digital PCR-Based Urinary Bladder Cancer Test by Year End

|url=https://www.genomeweb.com/molecular-diagnostics/norwegian-team-plans-debut-digital-pcr-based-urinary-bladder-cancer-test-year |access-date=24 July 2019}}{{cite journal | vauthors = Hiemcke-Jiwa LS, Minnema MC, Radersma-van Loon JH, Jiwa NM, de Boer M, Leguit RJ, de Weger RA, Huibers MM | display-authors = 6 | title = The use of droplet digital PCR in liquid biopsies: A highly sensitive technique for MYD88 p.(L265P) detection in cerebrospinal fluid | journal = Hematological Oncology | volume = 36 | issue = 2 | pages = 429–435 | date = April 2018 | pmid = 29210102 | doi = 10.1002/hon.2489 | s2cid = 4968214 | doi-access = free }} 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.{{cite news | vauthors = Paxton A | date = October 2017 |title= Revived hopes, fresh challenges with liquid biopsy

|url=https://www.captodayonline.com/revived-hopes-fresh-challenges-liquid-biopsy/ |access-date=24 July 2019}}{{cite news | vauthors = Bhadra K, Mellert H, Pestano G |date=5 Jun 2017 |title= Adoption of Liquid Biopsy Tests for NSCLC|url=http://www.clpmag.com/2017/06/adoption-liquid-biopsy-tests-nsclc/ |access-date=24 July 2019}} This reduced time to results has been used by physicians to expedite treatments tailored to biopsy 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.{{cite journal | vauthors = Sacher AG, Paweletz C, Dahlberg SE, Alden RS, O'Connell A, Feeney N, Mach SL, Jänne PA, Oxnard GR | display-authors = 6 | title = Prospective Validation of Rapid Plasma Genotyping for the Detection of EGFR and KRAS Mutations in Advanced Lung Cancer | journal = JAMA Oncology | volume = 2 | issue = 8 | pages = 1014–1022 | date = August 2016 | pmid = 27055085 | pmc = 4982795 | doi = 10.1001/jamaoncol.2016.0173 }} The application of liquid biopsy tests have also been studied in patients with breast,{{cite journal | vauthors = Olsson E, Winter C, George A, Chen Y, Howlin J, Tang MH, Dahlgren M, Schulz R, Grabau D, van Westen D, Fernö M, Ingvar C, Rose C, Bendahl PO, Rydén L, Borg Å, Gruvberger-Saal SK, Jernström H, Saal LH | display-authors = 6 | title = Serial monitoring of circulating tumor DNA in patients with primary breast cancer for detection of occult metastatic disease | journal = EMBO Molecular Medicine | volume = 7 | issue = 8 | pages = 1034–1047 | date = August 2015 | pmid = 25987569 | pmc = 4551342 | doi = 10.15252/emmm.201404913 }} colorectal,{{cite journal | vauthors = Carpinetti P, Donnard E, Bettoni F, Asprino P, Koyama F, Rozanski A, Sabbaga J, Habr-Gama A, Parmigiani RB, Galante PA, Perez RO, Camargo AA | display-authors = 6 | title = The use of personalized biomarkers and liquid biopsies to monitor treatment response and disease recurrence in locally advanced rectal cancer after neoadjuvant chemoradiation | journal = Oncotarget | volume = 6 | issue = 35 | pages = 38360–38371 | date = November 2015 | pmid = 26451609 | pmc = 4742005 | doi = 10.18632/oncotarget.5256 }}{{cite journal | vauthors = Reinert T, Schøler LV, Thomsen R, Tobiasen H, Vang S, Nordentoft I, Lamy P, Kannerup AS, Mortensen FV, Stribolt K, Hamilton-Dutoit S, Nielsen HJ, Laurberg S, Pallisgaard N, Pedersen JS, Ørntoft TF, Andersen CL | display-authors = 6 | title = Analysis of circulating tumour DNA to monitor disease burden following colorectal cancer surgery | journal = Gut | volume = 65 | issue = 4 | pages = 625–634 | date = April 2016 | pmid = 25654990 | doi = 10.1136/gutjnl-2014-308859 | doi-access = free }} gynecologic,{{cite journal | vauthors = Pereira E, Camacho-Vanegas O, Anand S, Sebra R, Catalina Camacho S, Garnar-Wortzel L, Nair N, Moshier E, Wooten M, Uzilov A, Chen R, Prasad-Hayes M, Zakashansky K, Beddoe AM, Schadt E, Dottino P, Martignetti JA | display-authors = 6 | title = Personalized Circulating Tumor DNA Biomarkers Dynamically Predict Treatment Response and Survival In Gynecologic Cancers | journal = PLOS ONE | volume = 10 | issue = 12 | pages = e0145754 | year = 2015 | pmid = 26717006 | pmc = 4696808 | doi = 10.1371/journal.pone.0145754 | doi-access = free | bibcode = 2015PLoSO..1045754P }} and bladder cancers{{cite journal | vauthors = Dahmcke CM, Steven KE, Larsen LK, Poulsen AL, Abdul-Al A, Dahl C, Guldberg P | title = A Prospective Blinded Evaluation of Urine-DNA Testing for Detection of Urothelial Bladder Carcinoma in Patients with Gross Hematuria | journal = European Urology | volume = 70 | issue = 6 | pages = 916–919 | date = December 2016 | pmid = 27417036 | doi = 10.1016/j.eururo.2016.06.035 }} to monitor both the disease load and the tumor’s response to treatment.

Gene expression and RNA quantification studies have benefited from the increased precision and absolute quantification of dPCR.{{cite journal | vauthors = Lindner L, Cayrou P, Jacquot S, Birling MC, Herault Y, Pavlovic G | title = Reliable and robust droplet digital PCR (ddPCR) and RT-ddPCR protocols for mouse studies | journal = Methods | volume = 191 | pages = 95–106 | date = July 2021 | pmid = 32721466 | doi = 10.1016/j.ymeth.2020.07.004 | s2cid = 220851187 }} 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.

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.{{cite journal | vauthors = Taylor SC, Carbonneau J, Shelton DN, Boivin G | title = Optimization of Droplet Digital PCR from RNA and DNA extracts with direct comparison to RT-qPCR: Clinical implications for quantification of Oseltamivir-resistant subpopulations | journal = Journal of Virological Methods | volume = 224 | pages = 58–66 | date = November 2015 | pmid = 26315318 | doi = 10.1016/j.jviromet.2015.08.014 | doi-access = free }} 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.{{cite journal | vauthors = Whale AS, Huggett JF, Tzonev S | title = Fundamentals of multiplexing with digital PCR | journal = Biomolecular Detection and Quantification | volume = 10 | pages = 15–23 | date = December 2016 | pmid = 27990345 | pmc = 5154634 | doi = 10.1016/j.bdq.2016.05.002 }} 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.{{cite journal | vauthors = Sun B, Tao L, Zheng YL | title = Simultaneous quantification of alternatively spliced transcripts in a single droplet digital PCR reaction | journal = BioTechniques | volume = 56 | issue = 6 | pages = 319–325 | date = June 2014 | pmid = 24924392 | doi = 10.2144/000114179 | doi-access = free }}

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.{{cite journal | vauthors = Valencia CA, Rhodenizer D, Bhide S, Chin E, Littlejohn MR, Keong LM, Rutkowski A, Bonnemann C, Hegde M | display-authors = 6 | title = Assessment of target enrichment platforms using massively parallel sequencing for the mutation detection for congenital muscular dystrophy | journal = The Journal of Molecular Diagnostics | volume = 14 | issue = 3 | pages = 233–246 | year = 2012 | pmid = 22426012 | pmc = 3349841 | doi = 10.1016/j.jmoldx.2012.01.009 }}{{cite journal | vauthors = Philippe J, Derhourhi M, Durand E, Vaillant E, Dechaume A, Rabearivelo I, Dhennin V, Vaxillaire M, De Graeve F, Sand O, Froguel P, Bonnefond A | display-authors = 6 | title = What Is the Best NGS Enrichment Method for the Molecular Diagnosis of Monogenic Diabetes and Obesity? | journal = PLOS ONE | volume = 10 | issue = 11 | pages = e0143373 | year = 2015 | pmid = 26599467 | pmc = 4657897 | doi = 10.1371/journal.pone.0143373 | doi-access = free | bibcode = 2015PLoSO..1043373P }} 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.{{cite journal | vauthors = Ouellet E, Foley JH, Conway EM, Haynes C | title = Hi-Fi SELEX: A High-Fidelity Digital-PCR Based Therapeutic Aptamer Discovery Platform | journal = Biotechnology and Bioengineering | volume = 112 | issue = 8 | pages = 1506–1522 | date = August 2015 | pmid = 25727321 | doi = 10.1002/bit.25581 | s2cid = 39450798 }} Partitioning can also allow for more robust measurements of telomerase activity from cell lysates.{{cite book | vauthors = Ludlow AT, Shelton D, Wright WE, Shay JW | chapter = DdTRAP: A Method for Sensitive and Precise Quantification of Telomerase Activity | title = Digital PCR | series = Methods in Molecular Biology | volume = 1768 | pages = 513–529 | year = 2018 | pmid = 29717462 | pmc = 6046637 | doi = 10.1007/978-1-4939-7778-9_29 | isbn = 978-1-4939-7776-5 }}{{cite journal | vauthors = Sayed ME, Slusher AL, Ludlow AT | title = Droplet Digital TRAP (ddTRAP): Adaptation of the Telomere Repeat Amplification Protocol to Droplet Digital Polymerase Chain Reaction | journal = Journal of Visualized Experiments | issue = 147 | date = May 2019 | pmid = 31107456 | doi = 10.3791/59550 | s2cid = 155519448 }} 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).{{cite news | vauthors = Stein RA |date=1 July 2019 |title= Single-Cell Sequencing Sifts through Multiple Omics |url=https://www.genengnews.com/topics/omics/single-cell-sequencing-sifts-through-multiple-omics/ |access-date=1 August 2019}}

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.{{cite book | vauthors = Wood-Bouwens CM, Ji HP | chapter = Single Color Multiplexed DDPCR Copy Number Measurements and Single Nucleotide Variant Genotyping | title = Digital PCR | series = Methods in Molecular Biology | volume = 1768 | pages = 323–333 | year = 2018 | pmid = 29717451 | doi = 10.1007/978-1-4939-7778-9_18 | isbn = 978-1-4939-7776-5 }}

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.Low, H., Chan, SJ., Soo, GH. et al. Clarity™ digital PCR system: a novel platform for absolute quantification of nucleic acids. Anal Bioanal Chem 409, 1869–1875 (2017). https://doi.org/10.1007/s00216-016-0131-7

dPCR rose out of an approach first published in 1988 by Cetus Corporation when researchers showed that a single copy of the β-globin gene could be detected and amplified by PCR.{{cite journal | vauthors = Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA | display-authors = 6 | title = Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase | journal = Science | volume = 239 | issue = 4839 | pages = 487–491 | date = January 1988 | pmid = 2448875 | doi = 10.1126/science.239.4839.487 | bibcode = 1988Sci...239..487S }}{{cite journal | vauthors = Morley AA | title = Digital PCR: A brief history | journal = Biomolecular Detection and Quantification | volume = 1 | issue = 1 | pages = 1–2 | date = September 2014 | pmid = 27920991 | pmc = 5129430 | doi = 10.1016/j.bdq.2014.06.001 }} 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.{{cite journal | vauthors = Rutsaert S, Bosman K, Trypsteen W, Nijhuis M, Vandekerckhove L | title = Digital PCR as a tool to measure HIV persistence | journal = Retrovirology | volume = 15 | issue = 1 | pages = 16 | date = January 2018 | pmid = 29378600 | pmc = 5789538 | doi = 10.1186/s12977-018-0399-0 | doi-access = free }} Alex Morley and Pamela Sykes formally established the method as a quantitative technique in 1992.{{cite journal | vauthors = Sykes PJ, Neoh SH, Brisco MJ, Hughes E, Condon J, Morley AA | title = Quantitation of targets for PCR by use of limiting dilution | journal = BioTechniques | volume = 13 | issue = 3 | pages = 444–449 | date = September 1992 | pmid = 1389177 }}

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.{{cite news | vauthors = Perkel J |date=11 April 2014 |title=The digital PCR revolution |url=https://www.science.org/content/article/digital-pcr-revolution |access-date=22 July 2019}} 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 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.{{cite journal | vauthors = Pohl G, Shih I | title = Principle and applications of digital PCR | journal = Expert Review of Molecular Diagnostics | volume = 4 | issue = 1 | pages = 41–47 | date = January 2004 | pmid = 14711348 | doi = 10.1586/14737159.4.1.41 | s2cid = 28271641 }}{{cite journal | vauthors = Dressman D, Yan H, Traverso G, Kinzler KW, Vogelstein B | title = Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 100 | issue = 15 | pages = 8817–8822 | date = July 2003 | pmid = 12857956 | pmc = 166396 | doi = 10.1073/pnas.1133470100 | doi-access = free | bibcode = 2003PNAS..100.8817D }}{{cite journal | vauthors = Diehl F, Li M, He Y, Kinzler KW, Vogelstein B, Dressman D | title = BEAMing: single-molecule PCR on microparticles in water-in-oil emulsions | journal = Nature Methods | volume = 3 | issue = 7 | pages = 551–559 | date = July 2006 | pmid = 16791214 | doi = 10.1038/nmeth898 | s2cid = 7059151 }} In 2007 Mikael Kubista, Stephen Bustin and coworkers published the first dPCR study using the first nanoliter platform, with a greater number of partitions, developed by Fluidigm, studying intracellular mRNA distribution.{{Cite journal |last=Sindelka |first=Radek |last2=Jonák |first2=Jiri |last3=Hands |first3=Rebecca |last4=Bustin |first4=Stephen A. |last5=Kubista |first5=Mikael |date=2008-02-01 |title=Intracellular expression profiles measured by real-time PCR tomography in the Xenopus laevis oocyte |url=https://academic.oup.com/nar/article/36/2/387/2409865 |journal=Nucleic Acids Research |volume=36 |issue=2 |pages=387–392 |doi=10.1093/nar/gkm1024 |issn=0305-1048|doi-access=free }}

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.{{cite news | vauthors = Butkus B |date=8 July 2010 |title=Digital PCR Space Heating Up as Life Science Tool Vendors Begin Staking Claims |url=https://www.genomeweb.com/pcr/digital-pcr-space-heating-life-science-tool-vendors-begin-staking-claims |access-date=22 July 2019}}{{cite book | vauthors = Ramakrishnan R, Qin J, Jones RC, Weaver LS | chapter = Integrated Fluidic Circuits (IFCs) for Digital PCR | title = Microfluidic Diagnostics | series = Methods in Molecular Biology | volume = 949 | pages = 423–431 | date = 2013 | pmid = 23329458 | doi = 10.1007/978-1-62703-134-9_27 | isbn = 978-1-62703-133-2 }}{{cite news | vauthors = Butkus B |date=29 Mar 2012 |title= RainDance Launches Digital PCR Platform; Claims Sensitivity, Operating Cost Superiority |url=https://www.genomeweb.com/pcr/raindance-launches-digital-pcr-platform-claims-sensitivity-operating-cost-superiority |access-date=22 July 2019}} In recent years, scientists have developed and commercialized dPCR-based diagnostics for several conditions, including non-small cell lung cancer and Down’s Syndrome.{{cite news |date=7 Apr 2016 |title= 'Liquid biopsy' blood test detects genetic mutations in common form of lung cancer |url=https://www.sciencedaily.com/releases/2016/04/160407115907.htm |access-date=22 July 2019}}{{cite news |date=2 Mar 2018 |title= Korea's BioCore First to Commercialize NIPT Based on Digital PCR |url=https://www.genomeweb.com/pcr/koreas-biocore-first-commercialize-nipt-based-digital-pcr#.XTYVM5NKh-V |access-date=22 July 2019}} The first dPCR system for clinical use was CE-marked in 2017 and cleared by the US Food and Drug Administration in 2019, for diagnosing chronic myeloid leukemia.{{cite news |date=5 Dec 2017 |title= Bio-Rad Gets First CE Mark on Clinical ddPCR Test |url=https://www.genomeweb.com/pcr/bio-rad-gets-first-ce-mark-clinical-ddpcr-test |access-date=22 Jul 2019}}

{{reflist|32em}}

• [https://www.scribd.com/doc/241232380/Digital-PCR-Protocol Digital PCR Protocol]
• [https://doi.org/10.1016/j.ddtec.2005.08.017 High Throughput, Nanoliter Quantitative PCR]
• [https://doi.org/10.1038/nmeth1007-869 PCR's next frontier]

{{Polymerase chain reaction}}

Category:Molecular biology

Category:Polymerase chain reaction

Category:Laboratory techniques