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Single molecule detection in nanofluidic digital array enables accurate measurement of DNA copy number

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Abstract

Digital polymerase chain reaction (PCR) is a promising technique for estimating target DNA copy number. PCR solution is distributed throughout numerous partitions, and following amplification, target DNA copy number is estimated based on the proportion of partitions containing amplified DNA. Here, we identify approaches for obtaining reliable digital PCR data. Single molecule amplification efficiency was significantly improved following fragmentation of total DNA and bias in copy number estimates reduced by analysis of short intact target DNA fragments. Random and independent distribution of target DNA molecules throughout partitions, which is critical to accurate digital PCR measurement, was demonstrated by spatial distribution analysis. The estimated relative uncertainty for target DNA concentration was under 6% when analyzing five digital panels comprising 765 partitions each, provided the panels contained an average of 212 to 3,365 template molecules. Partition volume was a major component of this uncertainty estimate. These findings can be applied to other digital PCR studies to improve confidence in such measurements.

Digital PCR amplification plot (left) and panel read out (right) of HindIII-digested pIRMM69. pIRMM69 contains one HindIII restriction enzyme site outside the hmg and transgene fragments used as targets in PCR. Red boxes with white shade denote positive hits containing one or more target DNA molecules, and white boxes with grey shade refer to no target being amplified.

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Abbreviations

bp:

Base pair

Ct:

Cycle threshold

DNA:

Deoxyribonucleic acid

ERM:

European reference material

GM:

Genetically modified

hmg:

High mobility group protein A

NTC:

No template control

PCR:

Polymerase chain reaction

References

  1. Iwao-Koizumi K, Maekawa K, Nakamura Y, Saito S, Kawamoto S, Nakagawara A, Kato K (2007) BMC Genomics 8:206

    Article  Google Scholar 

  2. Qin J, Jones RC, Ramakrishnan R (2008) Nucleic Acids Res 36:e116

    Article  Google Scholar 

  3. Vogelstein B, Kinzler KW (1999) Proc Natl Acad Sci USA 96:9236

    Article  CAS  Google Scholar 

  4. Pohl G, Shih Ie M (2004) Expert Rev Mol Diagn 4:41

    Article  CAS  Google Scholar 

  5. Oehler VG, Qin J, Ramakrishnan R, Facer G, Ananthnarayan S, Cummings C, Deininger M, Shah N, McCormick F, Willis S, Daridon A, Unger M, Radich JP (2009) Leukemia 23:396a

    Article  Google Scholar 

  6. Spurgeon SL, Jones RC, Ramakrishnan R (2008) PLoS ONE 3:e1662

    Article  Google Scholar 

  7. Lo YM, Chiu RW (2008) Clin Chem 54:461

    Article  CAS  Google Scholar 

  8. Lo YM, Lun FM, Chan KC, Tsui NB, Chong KC, Lau TK, Leung TY, Zee BC, Cantor CR, Chiu RW (2007) Proc Natl Acad Sci USA 104:13116

    Article  CAS  Google Scholar 

  9. Lun FM, Chiu RW, Allen Chan KC, Yeung Leung T, Kin Lau T, Dennis Lo YM (2008) Clin Chem 54:1664

    Article  CAS  Google Scholar 

  10. Weisenberger DJ, Trinh BN, Campan M, Sharma S, Long TI, Ananthnarayan S, Liang G, Esteva FJ, Hortobagyi GN, McCormick F, Jones PA, Laird PW (2008) Nucleic Acids Res 36:4689

    Article  CAS  Google Scholar 

  11. Warren L, Bryder D, Weissman IL, Quake SR (2006) Proc Natl Acad Sci USA 103:17807

    Article  CAS  Google Scholar 

  12. Corbisier P, Broeders S, Charels D, Trapmann S, Vincent S, Emons H (2007) EUR Report 22948–European Community, Luxembourg – ISBN 978-92-79-07139-3

  13. European Commission (2004) Off. J. Eur. Union L 348:18

  14. Dube S, Qin J, Ramakrishnan R (2008) PLoS ONE 3:e2876

    Article  Google Scholar 

  15. Ripley BD (1977) J R Stat Soc B 39:172

    Google Scholar 

  16. Baddeley A, Turner R (2005) J Stat Softw 12:1

    Google Scholar 

  17. Vazquez R, Steinberg ML (1999) Biotechniques 26:91

    CAS  Google Scholar 

  18. Georgiou CD, Papapostolou I (2006) Anal Biochem 358:247

    Article  CAS  Google Scholar 

  19. Stenman J, Orpana A (2001) Nat Biotechnol 19:1011

    Article  CAS  Google Scholar 

  20. Jarvius J, Melin J, Goransson J, Stenberg J, Fredriksson S, Gonzalez-Rey C, Bertilsson S, Nilsson M (2006) Nat Methods 3:725

    Article  CAS  Google Scholar 

  21. Blow N (2007) Nat Meth 4:869

    Article  CAS  Google Scholar 

Download references

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Authors and Affiliations

  1. National Measurement Institute, Lindfield, New South Wales, Australia

    Somanath Bhat, Jan Herrmann, Paul Armishaw, Philippe Corbisier & Kerry R. Emslie

  2. European Commission, Joint Research Centre, Institute for Reference Materials and Measurements (IRMM), Retieseweg 111, 2440, Geel, Belgium

    Philippe Corbisier

Authors
  1. Somanath Bhat
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  2. Jan Herrmann
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  3. Paul Armishaw
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  4. Philippe Corbisier
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  5. Kerry R. Emslie
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Correspondence to Kerry R. Emslie.

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Bhat, S., Herrmann, J., Armishaw, P. et al. Single molecule detection in nanofluidic digital array enables accurate measurement of DNA copy number. Anal Bioanal Chem 394, 457–467 (2009). https://doi.org/10.1007/s00216-009-2729-5

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  • DOI: https://doi.org/10.1007/s00216-009-2729-5

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