DNA Detection by Cascade Enzymatic Signal Amplification

  • Bingjie Zou
  • Yinjiao Ma
  • Guohua Zhou
Part of the Methods in Molecular Biology book series (MIMB, volume 1039)


Although many approaches based on template replication were developed and applied in DNA detection, cross-contamination from amplicons is always a vexing problem. Thus, signal amplification is preferable for DNA detection due to its low risk of cross-contamination from amplicons. Here, we proposed a cascade enzymatic signal amplification (termed as CESA) by coupling Afu flap endonuclease with nicking endonuclease, including three steps: invasive signal amplification, flap ligation, and nicking endonuclease signal amplification. Because of the advantages of low risk of contamination, no sequence requirement of target DNA, and the universal reaction conditions for any target detection, CESA has a great potential in clinical diagnosis.

Key words

Signal amplification Afu flap endonuclease Nicking endonuclease Molecular beacon 



This work was supported by the National Natural Science Foundation of China (Grant 20975113, 31200638, and 21275161).


  1. 1.
    Compton J (1991) Nucleic acid sequence-based amplification. Nature 350:91–92PubMedCrossRefGoogle Scholar
  2. 2.
    Lizardi PM, Huang X, Zhu Z, Bray-Ward P, Thomas DC, Ward DC (1998) Mutation detection and single-molecule counting using isothermal rolling-circle amplification. Nat Genet 19:225–232PubMedCrossRefGoogle Scholar
  3. 3.
    Mullis KB, Faloona FA (1987) Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol 155:335–350PubMedCrossRefGoogle Scholar
  4. 4.
    Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N et al (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28:E63PubMedCrossRefGoogle Scholar
  5. 5.
    Zou B, Ma Y, Wu H, Zhou G (2012) Signal amplification by rolling circle amplification on universal flaps yielded from target-specific invasive reaction. Analyst 137:729–734PubMedCrossRefGoogle Scholar
  6. 6.
    Schwartz JR, Sarvaiya PJ, Leiva LE, Velez MC, Singleton TC, Yu LC et al (2012) A facile, branched DNA assay to quantitatively measure glucocorticoid receptor auto-regulation in T-cell acute lymphoblastic leukemia. Chin J Cancer 31:381–391PubMedCrossRefGoogle Scholar
  7. 7.
    Baumeister MA, Zhang N, Beas H, Brooks JR, Canchola JA, Cosenza C et al (2012) A sensitive branched DNA HIV-1 signal amplification viral load assay with single day turnaround. PLoS One 7:e33295PubMedCrossRefGoogle Scholar
  8. 8.
    Zou B, Ma Y, Wu H, Zhou G (2011) Ultrasensitive DNA detection by cascade enzymatic signal amplification based on Afu flap endonuclease coupled with nicking endonuclease. Angew Chem Int Ed Engl 50:7395–7398PubMedCrossRefGoogle Scholar
  9. 9.
    Kaiser MW, Lyamicheva N, Ma W, Miller C, Neri B, Fors L et al (1999) A comparison of eubacterial and archaeal structure-specific 5′-exonucleases. J Biol Chem 274:21387–21394PubMedCrossRefGoogle Scholar
  10. 10.
    Zhu Z, Samuelson JC, Zhou J, Dore A, Xu SY (2004) Engineering strand-specific DNA nicking enzymes from the type IIS restriction endonucleases BsaI, BsmBI, and BsmAI. J Mol Biol 337:573–583PubMedCrossRefGoogle Scholar
  11. 11.
    Hall JG, Eis PS, Law SM, Reynaldo LP, Prudent JR, Marshall DJ et al (2000) Sensitive detection of DNA polymorphisms by the serial invasive signal amplification reaction. Proc Natl Acad Sci U S A 97:8272–8277PubMedCrossRefGoogle Scholar
  12. 12.
    Lyamichev V, Brow MA, Dahlberg JE (1993) Structure-specific endonucleolytic cleavage of nucleic acids by eubacterial DNA polymerases. Science 260:778–783PubMedCrossRefGoogle Scholar
  13. 13.
    Lyamichev V, Mast AL, Hall JG, Prudent JR, Kaiser MW, Takova T et al (1999) Polymorphism identification and quantitative detection of genomic DNA by invasive cleavage of oligonucleotide probes. Nat Biotechnol 17:292–296PubMedCrossRefGoogle Scholar
  14. 14.
    Kiesling T, Cox K, Davidson EA, Dretchen K, Grater G, Hibbard S et al (2007) Sequence specific detection of DNA using nicking endonuclease signal amplification (NESA). Nucleic Acids Res 35:e117PubMedCrossRefGoogle Scholar
  15. 15.
    Li JJ, Chu Y, Lee BY, Xie XS (2008) Enzymatic signal amplification of molecular beacons for sensitive DNA detection. Nucleic Acids Res 36:e36PubMedCrossRefGoogle Scholar
  16. 16.
    Tang Z, Wang K, Tan W, Li J, Liu L, Guo Q et al (2003) Real-time monitoring of nucleic acid ligation in homogenous solutions using molecular beacons. Nucleic Acids Res 31:e148PubMedCrossRefGoogle Scholar
  17. 17.
    Allawi HT, SantaLucia J Jr (1997) Thermodynamics and NMR of internal G.T mismatches in DNA. Biochemistry 36:10581–10594PubMedCrossRefGoogle Scholar
  18. 18.
    SantaLucia J Jr (1998) A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc Natl Acad Sci U S A 95:1460–1465PubMedCrossRefGoogle Scholar
  19. 19.
    Lyamichev VI, Kaiser MW, Lyamicheva NE, Vologodskii AV, Hall JG, Ma WP et al (2000) Experimental and theoretical analysis of the invasive signal amplification reaction. Biochemistry 39:9523–9532PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, New York 2013

Authors and Affiliations

  • Bingjie Zou
    • 1
  • Yinjiao Ma
    • 1
  • Guohua Zhou
    • 1
  1. 1.Department of PharmacologyJinling Hospital, Nanjing University School of MedicineNanjingChina

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