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Entering the Pantheon of 21st Century Molecular Biology Tools: A Perspective on Digital PCR

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Digital PCR

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1768))

Abstract

After several decades of relatively modest use, in the last several years digital PCR (dPCR) has grown to become the new gold standard for nucleic acid quantification. This coincides with the commercial availability of scalable, affordable, and reproducible droplet-based dPCR platforms in the past five years and has led to its rapid dissemination into diverse research fields and testing applications. Among these, it has been adopted most vigorously into clinical oncology where it is beginning to be used for plasma genotyping in cancer patients undergoing treatment. Additionally, innovation across the scientific community has extended the benefits of reaction partitioning beyond DNA and RNA quantification alone, and demonstrated its usefulness in evaluating DNA size and integrity, the physical linkage of colocalized markers, levels of enzyme activity and specific cation concentrations in a sample, and more. As dPCR technology gains in popularity and breadth, its power and simplicity can often be taken for granted; thus, the reader is reminded that due diligence must be exercised in order to make claims not only of precision but also of accuracy in their measurements.

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References

  1. Hindson BJ, Ness KD, Masquelier DA, Belgrader P, Heredia NJ, Makarewicz AJ, Bright IJ, Lucero MY, Hiddessen AL, Legler TC, Kitano TK, Hodel MR, Petersen JF, Wyatt PW, Steenblock ER, Shah PH, Bousse LJ, Troup CB, Mellen JC, Wittmann DK, Erndt NG, Cauley TH, Koehler RT, So AP, Dube S, Rose KA, Montesclaros L, Wang S, Stumbo DP, Hodges SP, Romine S, Milanovich FP, White HE, Regan JF, Karlin-Neumann GA, Hindson CM, Saxonov S, Colston BW (2011) High-Throughput Droplet Digital PCR System for Absolute Quantitation of DNA Copy Number. Anal Chem 83(22):8604–8610. https://doi.org/10.1021/ac202028g

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Baker M (2012) Digital PCR hits its stride. Nat Methods 9(6):541–544. https://doi.org/10.1038/nmeth.2027

    Article  CAS  Google Scholar 

  3. Miliaras N (2014) Digital PCR comes of age. Genetic Engineering & Biotechnology News 34(4):14–16. https://doi.org/10.1089/gen.34.04.08

    Article  Google Scholar 

  4. Perkel J (2014) The digital PCR revolution. Science 344(6180):212–214. https://doi.org/10.1126/science.344.6180.212

    Article  Google Scholar 

  5. Pinheiro & Emslie, Chapter 2, this MIMB volume

    Google Scholar 

  6. Sacher AG, Paweletz C, Dahlberg SE, Alden RS, O'Connell A, Feeney N, Mach SL, Jänne PA, Oxnard GR (2016) Prospective validation of rapid plasma genotyping for the detection of EGFR and KRAS mutations in advanced lung cancer. JAMA Oncol 2(8):1014–1022. https://doi.org/10.1001/jamaoncol.2016.0173

    Article  PubMed  PubMed Central  Google Scholar 

  7. Mellert H, Foreman T, Jackson L, Maar D, Thurston S, Koch K, Weaver A, Cooper S, Dupuis N, Sathyanarayana UG, Greer J, Hahn W, Shelton D, Stonemetz P, Pestano GA (2017) Development and clinical utility of a blood-based test service for the rapid identification of actionable mutations in non-small cell lung carcinoma. J Mol Diagn 19(3):404–416. https://doi.org/10.1016/j.jmoldx.2016.11.004

    Article  CAS  PubMed  Google Scholar 

  8. Whale AS, Huggett JF, Cowen S, Speirs V, Shaw J, Ellison S, Foy CA, Scott DJ (2012) Comparison of microfluidic digital PCR and conventional quantitative PCR for measuring copy number variation. Nucleic Acids Res 40(11):e82. https://doi.org/10.1093/nar/gks203

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Hindson CM, Chevillet JR, Briggs HA, Gallichotte EN, Ruf IK, Hindson BJ, Vessella RL, Tewari M (2013) Absolute quantification by droplet digital PCR versus analog real-time PCR. Nat Methods 10(10):1003–1005. https://doi.org/10.1038/nmeth.2633

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Whale AS, Devonshire AS, Karlin-Neumann G, Regan J, Javier L, Cowen S, Fernandez-Gonzalez A, Jones G, Redshaw N, Beck J, Berger A, Combaret V, Kjersgaard ND, Davis L, Fina F, Forshew F, Andersen RF, Galbiati S, Hernańdez AG, Haynes CA, Janku F, Lacave R, Lee J, Mistry V, Pender A, Pradines A, Proudhon C, Saal L, Stieglitz E, Ulrich B, Foy CA, Parkes H, Tzonev S, Huggett JF (2017) International interlaboratory digital pcr study demonstrating high reproducibility for the measurement of a rare sequence variant. Anal Chem 89(3):1724–1733. https://doi.org/10.1021/acs.analchem.6b03980

    Article  CAS  PubMed  Google Scholar 

  11. Cao Y, Raith MR, Griffith JF (2015) Droplet digital PCR for simultaneous quantification of general and human-associated fecal indicators for water quality assessment. Water Res 70:337–349. https://doi.org/10.1016/j.watres.2014.12.008

    Article  CAS  PubMed  Google Scholar 

  12. Nadauld L, Regan JF, Miotke L, Pai RK, Longacre TA, Kwok SS, Saxonov S, Ford JM, Ji HP (2012) Quantitative and sensitive detection of cancer genome amplifications from formalin fixed paraffin embedded tumors with droplet digital PCR. Transl Med (Sunnyvale) 2(2):1–12. https://doi.org/10.4172/2161-1025.1000107

    Article  Google Scholar 

  13. Belgrader P, Tanner SC, Regan JF, Koehler R, Hindson BJ, Brown AS (2013) Droplet digital PCR measurement of HER2 copy number alteration in formalin-fixed paraffin-embedded breast carcinoma tissue. Clin Chem 59(6):991–994. https://doi.org/10.1373/clinchem.2012.197855

    Article  CAS  PubMed  Google Scholar 

  14. Dingle TC, Sedlak RH, Cook L, Jerome KR (2013) Tolerance of droplet-digital PCR vs real-time quantitative PCR to inhibitory substances. Clin Chem 59(11):1670–1672. https://doi.org/10.1373/clinchem.2013.211045

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Oxnard GR, Paweletz CP, Kuang Y, Mach SL, O'Connell A, Messineo MM, Luke JJ, Butaney M, Kirschmeier P, Jackman DM, Jänne PA (2014) Noninvasive detection of response and resistance in EGFR-mutant lung cancer using quantitative next-generation genotyping of cell-free plasma DNA. Clin Cancer Res 20(6):1698–1705. https://doi.org/10.1158/1078-0432.CCR-13-2482

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Garcia-Murillas I, Schiavon G, Weigelt B, Ng C, Hrebien S, Cutts RJ, Cheang M, Osin P, Nerurkar A, Kozarewa GJA, Dowsett M, Reis-Filho JS, Smith IE, Turner NC (2015) Mutation tracking in circulating tumor DNA predicts relapse in early breast cancer. Sci Transl Med 7(302):302ra133. https://doi.org/10.1126/scitranslmed.aab0021

    Article  PubMed  Google Scholar 

  17. Janku F, Huang HJ, Fujii T, Shelton DN, Madwani K, Fu S, Tsimberidou AM, Piha-Paul SA, Wheler JJ, Zinner RG, Naing A, Hong DS, Karp DD, Cabrilo G, Kopetz ES, Subbiah V, Luthra R, Kee BK, Eng C, Morris VK, Karlin-Neumann GA, Meric-Bernstam F (2017) Multiplex KRASG12/G13 mutation testing of unamplified cell-free DNA from the plasma of patients with advanced cancers using droplet digital polymerase chain reaction. Ann Oncol 28(3):642–650. https://doi.org/10.1093/annonc/mdw670

    Article  CAS  PubMed  Google Scholar 

  18. Strain MC, Lada SM, Luong T, Rought SE, Gianella S, Terry VH, Spina CA, Woelk CH, Richman DD (2013) Highly precise measurement of HIV DNA by droplet digital PCR. PLoS One 8(4):e55943. https://doi.org/10.1371/journal.pone.0055943

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Persaud D, Gay H, Ziemniak C, Chen YH, Piatak M Jr, Chun TW, Strain M, Richman D, Luzuriaga K (2013) Absence of detectable HIV-1 viremia after treatment cessation in an infant. N Engl J Med 369(19):1828–1835. https://doi.org/10.1056/NEJMoa1302976

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Leibovitch EC, Brunetto GS, Caruso B, Fenton K, Ohayon J, Reich DS, Jacobson S (2014) Coinfection of human herpesviruses 6A (HHV-6A) and HHV-6B as demonstrated by novel digital droplet PCR assay. PLoS One 9(3):e92328. https://doi.org/10.1371/journal.pone.0092328

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Abyzov A, Mariani J, Palejev D, Zhang Y, Haney MS, Tomasini L, Ferrandino AF, Rosenberg Belmaker LA, Szekely A, Wilson M, Kocabas A, Calixto NE, Grigorenko EL, Huttner A, Chawarska K, Weissman S, Urban AE, Gerstein M, Vaccarino FM (2012) Somatic copy number mosaicism in human skin revealed by induced pluripotent stem cells. Nature 492(7429):438–442. https://doi.org/10.1038/nature11629

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Handsaker RE, Van Doren V, Berman JR, Genovese G, Kashin S, Boettger LM, McCarroll SA (2015) Large multiallelic copy number variations in humans. Nat Genet 47(3):296–303. https://doi.org/10.1038/ng.3200

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Davis JM, Searles VB, Anderson N, Keeney J, Dumas L, Sikela JM (2014) DUF1220 dosage is linearly associated with increasing severity of the three primary symptoms of autism. PLoS Genet 10(3):e1004241. https://doi.org/10.1371/journal.pgen.1004241

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. 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 (2015) Noninvasive detection of activating estrogen receptor 1 (ESR1) mutations in estrogen receptor-positive metastatic breast cancer. Clin Chem 61(7):974–982. https://doi.org/10.1373/clinchem.2015.238717

    Article  CAS  PubMed  Google Scholar 

  25. Thress KS, Brant R, Carr TH, Dearden S, Jenkins S, Brown H, Hammett T, Cantarini M, Barrett JC (2015) EGFR mutation detection in ctDNA from NSCLC patient plasma: A cross-platform comparison of leading technologies to support the clinical development of AZD9291. Lung Cancer 90(3):509–515. https://doi.org/10.1016/j.lungcan.2015.10.004

    Article  PubMed  Google Scholar 

  26. Gu J, Zang W, Liu B, Li L, Huang L, Li S, Rao G, Yu Y, Zhou Y (2017) Evaluation of digital PCR for detecting low-level EGFR mutations in advanced lung adenocarcinoma patients: a cross-platform comparison study. Oncotarget. https://doi.org/10.18632/oncotarget.18866. [Epub ahead of print]

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

    Article  CAS  PubMed  Google Scholar 

  28. Aldhous MC, Abu Bakar S, Prescott NJ, Palla R, Soo K, Mansfield JC, Mathew CG, Satsangi J, Armour JA (2010) Measurement methods and accuracy in copy number variation: failure to replicate associations of beta-defensin copy number with Crohn's disease. Hum Mol Genet 19(24):4930–4938. https://doi.org/10.1093/hmg/ddq411

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Liu S, Yao L, Ding D, Zhu H (2010) CCL3L1 copy number variation and susceptibility to HIV-1 infection: a meta-analysis. PLoS One 5(12):e15778. https://doi.org/10.1371/journal.pone.0015778

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. 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 M1, Boehnke M, Altshuler DM, Frayling TM, Hirschhorn JN, McCarroll SA (2015) Structural forms of the human amylase locus and their relationships to SNPs, haplotypes and obesity. Nat Genet 47(8):921–925. https://doi.org/10.1038/ng.3340

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Albayrak C, Jordi CA, Zechner C, Lin J, Bichsel CA, Khammash M, Tay S (2016) Digital quantification of proteins and mRNA in single mammalian cells. Mol Cell 61(6):914–924. https://doi.org/10.1016/j.molcel.2016.02.030

    Article  CAS  PubMed  Google Scholar 

  32. Cheng N, Zhu P, Xu Y, Huang K, Luo Y, Yang Z, Xu W (2016) High-sensitivity assay for Hg (II) and Ag (I) ion detection: A new class of droplet digital PCR logic gates for an intelligent DNA calculator. Biosens Bioelectron 84:1–6. https://doi.org/10.1016/j.bios.2016.04.084

    Article  CAS  PubMed  Google Scholar 

  33. Shen F (2017) SlipChip device for digital nucleic acid amplification. Methods Mol Biol 1547:123–132. https://doi.org/10.1007/978-1-4939-6734-6_10

    Article  PubMed  Google Scholar 

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Authors and Affiliations

  1. Digital Biology Center, Bio-Rad Laboratories, Pleasanton, CA, USA

    George Karlin-Neumann & Francisco Bizouarn

Authors
  1. George Karlin-Neumann
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  2. Francisco Bizouarn
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Correspondence to George Karlin-Neumann .

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Editors and Affiliations

  1. Digital Biology Center, Bio-Rad Laboratories, Pleasanton, California, USA

    George Karlin-Neumann

  2. Digital Biology Center, Bio-Rad Laboratories, Pleasanton, California, USA

    Francisco Bizouarn

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Karlin-Neumann, G., Bizouarn, F. (2018). Entering the Pantheon of 21st Century Molecular Biology Tools: A Perspective on Digital PCR. In: Karlin-Neumann, G., Bizouarn, F. (eds) Digital PCR. Methods in Molecular Biology, vol 1768. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7778-9_1

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  • DOI: https://doi.org/10.1007/978-1-4939-7778-9_1

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7776-5

  • Online ISBN: 978-1-4939-7778-9

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