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Agarose Droplet Microfluidics for Highly Parallel and Efficient Single Molecule Emulsion PCR

  • Xuefei Leng
  • Chaoyong James YangEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 949)

Abstract

Agarose emulsion droplet microfluidic technology for single copy emulsion PCR (ePCR) is a suitable technique for the detection of single copy DNA molecules. It improves the traditional ePCR by employing agarose with low melting and low gelling temperatures, which is coupled with PCR forward primers using Schiff-base reaction. Highly uniform monodisperse nanoliter agarose droplets each containing PCR reagents and single copy template are produced with a microfabricated emulsion generator. Following PCR, the cooled droplets transform to microbeads carrying amplicons to maintain the monocolonity of each droplet, which can be further analyzed. This method allows high-throughput generation of uniform droplets and enables high PCR efficiency, making it a promising platform for many single copy genetic studies.

Key words

Emulsion PCR Microfluidics Bioconjugation Droplet Genetic analysis 

Notes

Acknowledgments

This work was supported by National Scientific Foundation of China (20805038, 20620130427) and National Basic Research Program of China (2007CB935603, 2010CB732402).

References

  1. 1.
    Dressman D, Yan H, Traverso G, Kinzler KW, Vogelstein B (2003) Proc Natl Acad Sci USA 100:8817CrossRefGoogle Scholar
  2. 2.
    Kelly BT, Baret JC, Taly V, Griffiths AD (2007) Chem Commun 1773.Google Scholar
  3. 3.
    Leamon JH, Link DR, Egholm M, Rothberg JM (2006) Nat Methods 3:541CrossRefGoogle Scholar
  4. 4.
    Li M, Chen WD, Papadopoulos N, Goodman SN, Bjerregaard NC, Laurberg S, Levin B, Juhl H, Arber N, Moinova H, Durkee K, Schmidt K, He YP, Diehl F, Velculescu VE, Zhou SB, Diaz LA, Kinzler KW, Markowitz SD, Vogelstein B (2009) Nat Biotechnol 27:858CrossRefGoogle Scholar
  5. 5.
    Li M, Diehl F, Dressman D, Vogelstein B, Kinzler KW (2006) Nat Methods 3:95CrossRefGoogle Scholar
  6. 6.
    Wheeler DA, Srinivasan M, Egholm M et al (2008) Nature 452:872CrossRefGoogle Scholar
  7. 7.
    Shendure J, Porreca GJ, Reppas NB, Lin XX, McCutcheon JP, Rosenbaum AM, Wang MD, Zhang K, Mitra RD, Church GM (2005) Science 309:1728CrossRefGoogle Scholar
  8. 8.
    Nakano M, Komatsu J, Matsuura S, Takashima K, Katsura S, Mizuno A (2003) J Biotechnol 102:117CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media,LLC 2013

Authors and Affiliations

  1. 1.Department of Chemical Biology, College of Chemistry and Chemical EngineeringXiamen UniversityXiamenPeople’s Republic of China

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