Helicase‐Dependent Amplification of Nucleic Acids

Yun Cao1, Hyun‐Jin Kim1, Ying Li1, Huimin Kong1, Bertrand Lemieux1

1 BioHelix Corporation, Beverly, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 15.11
DOI:  10.1002/0471142727.mb1511s104
Online Posting Date:  October, 2013
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Abstract

Helicase‐dependent amplification (HDA) is a novel method for the isothermal in vitro amplification of nucleic acids. The HDA reaction selectively amplifies a target sequence by extension of two oligonucleotide primers. Unlike the polymerase chain reaction (PCR), HDA uses a helicase enzyme to separate the deoxyribonucleic acid (DNA) strands, rather than heat denaturation. This allows DNA amplification without the need for thermal cycling. The helicase used in HDA is a helicase super family II protein obtained from a thermophilic organism, Thermoanaerobacter tengcongensis (TteUvrD). This thermostable helicase is capable of unwinding blunt‐end nucleic acid substrates at elevated temperatures (60° to 65°C). The HDA reaction can also be coupled with reverse transcription for ribonucleic acid (RNA) amplification. The products of this reaction can be detected during the reaction using fluorescent probes when incubations are conducted in a fluorimeter. Alternatively, products can be detected after amplification using a disposable amplicon containment device that contains an embedded lateral flow strip. Curr. Protoc. Mol. Biol. 104:15.11.1‐15.11.12. © 2013 by John Wiley & Sons, Inc.

Keywords: isothermal amplification; nucleic acid quantitation; lateral flow strip

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Helicase‐Dependent Amplification
  • Alternate Protocol 1: qHDA Real‐Time Quantitative Helicase‐Dependent Amplification
  • Alternate Protocol 2: Reverse Transcriptase Helicase‐Dependent Amplification (RT‐HDA)
  • Alternate Protocol 3: Asymmetric HDA
  • Support Protocol 1: Primer Design Guidelines
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Helicase‐Dependent Amplification

  Materials
  • IsoAmp II and IsoAmp III Universal tHDA kits (BioHelix) containing:
    • IsoAmp II or IsoAmp III enzyme mix (50 reactions)
    • IsoAmp dNTP solution (50 reactions)
    • 10× annealing buffer II
    • 100 mM MgSO 4
    • 500 mM NaCl
    • 1 ng/µl pCNG1 (control template)
    • 5 µM NGF3 (forward primer)
    • 5 µM NGR3 (reverse primer)
  • Milli‐Q water
  • DNA template
  • Forward and reverse primers (5 µM each; see Support Protocol)
  • Mineral oil
  • 0.2‐ml or 0.5‐ml microcentrifuge tubes
  • Sterile filter tips
  • Vortex
  • Thermal cycler, heating block, water bath, or incubator at 65°C
  • Additional reagents and equipment for 2% agarose gel electrophoresis (unit 2.5)

Alternate Protocol 1: qHDA Real‐Time Quantitative Helicase‐Dependent Amplification

  Additional Materials (also see protocol 1Basic Protocol)
  • EvaGreen (Biotium, cat. no. 31000)
  • ROX reference dye (Invitrogen, cat. no. 12223‐012)
  • 0.2‐ml optically clear tubes (ABI)
  • 7300 Real‐Time PCR System (ABI)

Alternate Protocol 2: Reverse Transcriptase Helicase‐Dependent Amplification (RT‐HDA)

  Additional Materials (also see protocol 1Basic Protocol)
  • 15 U/µl reverse transcriptase (ThermoScript RT, Invitrogen, cat. no. 12236‐022)

Alternate Protocol 3: Asymmetric HDA

  Additional Materials (also see protocol 1Basic Protocol)
  • BESt cassette kit (BioHelix, cat. no. D1100S)
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Figures

Videos

Literature Cited

  An, L., Tang, W., Ranalli, T.A., Kim, H.J., Wytiaz, J., and Kong, H. 2005. Characterization of a thermostable UvrD helicase and its participation in helicase‐dependent amplification. J. Biol. Chem. 280:28952‐28958.
  Chow, W.H., McCloskey, C., Tong, Y., Hu, L., You, Q., Kelly, C.P., Kong, H., Tang, Y.W., and Tang, W. 2008. Application of isothermal helicase‐dependent amplification with a disposable detection device in a simple sensitive stool test for toxigenic Clostridium difficile. J. Mol. Diagn. 10:452‐458.
  Goldmeyer, J., Kong, H., and Tang, W. 2007. Development of a novel one‐tube isothermal reverse transcription thermophilic helicase‐dependent amplification platform for rapid RNA detection. J. Mol. Diagn. 9:639‐644.
  Goldmeyer, J., Li, H., McCormac, M., Cook, S., Stratton, C., Lemieux, B., Kong, H., Tang, W., and Tang, Y.W. 2008. Identification of Staphylococcus aureus and determination of methicillin resistance directly from positive blood cultures by isothermal amplification and disposable detection device. J. Clin. Microbiol. 46:1534‐1536.
  Kim, H.J., Tong, Y., Tang, W., Quimson, L., Cope, V.A., Pan, X., Motre, A., Kong, R., Hong, J., Kohn, D., Miller, N.S., Poulter, M.D., Kong, H., Tang, Y.W., and Yen‐Lieberman, B. 2011. A rapid and simple isothermal nucleic acid amplification test for detection of herpes simplex virus types 1 and 2. J. Clin. Virol. 50:26‐30.
  Li, Y., Jortani, S.A., Ramey‐Hartung, B., Hudson, E., Lemieux, B., and Kong, H. 2011. Genotyping three SNPs affecting warfarin drug response by isothermal real‐time HDA assays. Clin. Chim. Acta 412:79‐85.
  Mao, F., Leung, W.Y., and Xin, X. 2007. Characterization of EvaGreen and the implication of its physicochemical properties for qPCR applications. BMC Biotechnol. 7:76.
  Motré, A., Kong, R., and Li, Y. 2011. Improving isothermal DNA amplification speed for the rapid detection of Mycobacterium tuberculosis. J. Microbiol. Methods 84:343‐345.
  Patel, J.C., Oberstaller, J., Xayavong, M., Narayanan, J., Debarry, J.D., Srinivasamoorthy, G., Villegas, L., Escalante, A.A., Dasilva, A., Peterson, D.S., Barnwell, J.W., Kissinger, J.C., Udhayakumar, V., and Lucchi, N.W. 2013. Real‐Time Loop‐Mediated Isothermal Amplification (RealAmp) for the species‐specific identification of plasmodium vivax. PLoS One 8: e54986.
  Tang, W., Chow, W.H.A., Li, Y., Kong, H., Tang, Y‐W., and Lemieux, B. 2010. Nucleic acid assay system for tier II laboratories and moderately complex clinics to detect HIV in low‐resource settings. J. Infect. Dis. 201:S46‐S51.
  Tong, Y., Tang, W., Kim, H.J., Pan, X., Ranalli, T.A., and Kong, H. 2008. Development of isothermal TaqMan assays for detection of biothreat organisms. BioTechniques 45:543‐557.
  Tong, Y., Lemieux, B., and Kong, H. 2011. Multiple strategies to improve sensitivity, speed and robustness of isothermal nucleic acid amplification for rapid pathogen detection. BMC Biotechnol. 11:50.
  Tong, Y., McCarthy, K., Kong, H., and Lemieux, B. 2012. Development and comparison of a rapid isothermal nucleic acid amplification test for typing of herpes simplex virus types 1 and 2 on a portable fluorescence detector. J. Mol. Diagn. 14:569‐576.
Internet Resources
  http://www.biohelix.com
  Provides information on HDA technology and reagents.
  http://www.idtdna.com/Scitools/Applications/Primerquest/Advanced.aspx
  Provides primer design services.
  http://primer3.wi.mit.edu/
  Provides primer design services.
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