BeadCons: Detection of Nucleic Acid Sequences by Flow Cytometry

Douglas Horejsh1, Federico Martini1, Maria Rosaria Capobianchi1

1 National Institute for Infectious Diseases “L. Spallazani”‐IRCSS, Rome, Italy
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 13.5
DOI:  10.1002/0471142956.cy1305s34
Online Posting Date:  November, 2005
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Abstract

Molecular beacons are single‐stranded nucleic acid structures with a terminal fluorophore and a distal, terminal quencher. These molecules are typically used in real‐time PCR assays, but have also been conjugated with solid matrices. This unit describes protocols related to molecular beacon–conjugated beads (BeadCons), whose specific hybridization with complementary target sequences can be resolved by cytometry. Assay sensitivity is achieved through the concentration of fluorescence signal on discrete particles. By using molecular beacons with different fluorophores and microspheres of different sizes, it is possible to construct a fluid array system with each bead corresponding to a specific target nucleic acid. Methods are presented for the design, construction, and use of BeadCons for the specific, multiplexed detection of unlabeled nucleic acids in solution. The use of bead‐based detection methods will likely lead to the design of new multiplex molecular diagnostic tools.

Keywords: BeadCons; molecular beacons; microspheres; flow cytometry; differential diagnosis

     
 
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Table of Contents

  • Basic Protocol 1: Construction of Beadcons Using a Direct Conjugation Between Biotinylated Molecular Beacons and Streptavidin‐Coated Microspheres
  • Alternate Protocol 1: Construction of Beadcons Using a Streptavidin Bridge Between Biotinylated Molecular Beacons and Biotin‐Coated Microspheres
  • Alternate Protocol 2: Detection of Asymmetric PCR Products using BeadCons
  • Alternate Protocol 3: Detection of Nucleic Acid Sequences in Multiplex using a Beadcons Array
  • Support Protocol 1: Design of Molecular Beacons for use in a Beadcons Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Construction of Beadcons Using a Direct Conjugation Between Biotinylated Molecular Beacons and Streptavidin‐Coated Microspheres

  Materials
  • 10 µM biotinylated molecular beacon (see recipe; see protocol 5 for design)
  • Sheath fluid: phosphate‐buffered saline (PBS), pH 7.4 ( appendix 2A)
  • 0.5% (w/v) streptavidin (SA)‐coated polystyrene microspheres (Bangs Laboratories, Polysciences, or G.Kisker GbR, http://www.kisker‐biotech.com) in PBS, pH 7.4 ( appendix 2A)
  • 100 µM test nucleic acids and controls (complementary oligos)
  • 13 × 100–mm polystyrene culture tubes
  • Centrifuge
  • Flow cytometer with filters suitable for collection of emission from the fluorochrome tag on the molecular beacon

Alternate Protocol 1: Construction of Beadcons Using a Streptavidin Bridge Between Biotinylated Molecular Beacons and Biotin‐Coated Microspheres

  • 5 mg/ml streptavidin (SA; Biosource International, Pierce Biotechnology, or Roche Applied Science); store up to 6 months at 4°C
  • 0.5% (w/v) biotin‐coated microspheres (Bangs Laboratories, Polysciences, or G.Kisker GbR, http://www.kisker‐biotech.com)

Alternate Protocol 2: Detection of Asymmetric PCR Products using BeadCons

  • DNA‐ or RNA‐based pathogen or test sample
  • DNA extraction kit (optional; e.g., Qiagen or Invitrogen)
  • 25 pmol oligo dT12‐18 primer (e.g., New England Biolabs)
  • DEPC‐treated H 2O (Gilman, )
  • 40 mM dNTP mix (10 mM of each dNTP; e.g., Promega)
  • M‐MLV reverse transcriptase (supplied with 5 × M‐MLV reaction buffer; e.g., Sigma‐Aldrich)
  • RNase inhibitor (e.g., Applied Biosystems)
  • 5 U/µl Taq DNA polymerase (supplied with 10 × Tag reaction buffer; e.g., Epicentre Technologies)
  • 25 mM MgCl 2
  • Oligonucleotide PCR primers (target sequence–specific)
  • BeadCons recognizing (−) strands of sequences of interest (see protocol 1 or protocol 2)
  • Microcentrifuge tubes or PCR tubes
  • Thermal cycler
  • Additional reagents and equipment for DNA extraction (Moore and Dowhan, )
  • NOTE: Special precautions must be used when working with RNA to avoid contamination with exogenous RNases. Refer to product inserts included with reverse transcription reagents, or see Chapter 4 of Ausubel et al. ( ) for further information.

Alternate Protocol 3: Detection of Nucleic Acid Sequences in Multiplex using a Beadcons Array

  • BeadCons (each target identified by a fluorescent dye and bead size combination; see protocol 1 or protocol 2)
  • 1 µM template nucleic acids, test samples and controls (see protocol 3 for preparation)

Support Protocol 1: Design of Molecular Beacons for use in a Beadcons Assay

  Materials
  • Molecular beacon design software (e.g., Beacon Designer 4.0; Premier Biosoft International, http://www.premierbiosoft.com)
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Figures

Videos

Literature Cited

Literature Cited
   Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (eds.) 2005. Current Protocols in Molecular Biology. John Wiley & Sons, Hoboken, N.J.
   Bonnet, G., Tyagi, S., Libchaber, A., and Kramer, F.R. 1999. Thermodynamic basis of the enhanced specificity of structured DNA probes. Proc. Natl. Acad. Sci. U.S.A. 96:6171‐6176.
   Drosten, C., Gunther, S., Preiser, W., van der Werf, S., Brodt, H.R., Becker, S., Rabenau, H., Panning, M., Kolesnikova, L., Fouchier, R.A., Berger, A., Burguiere, A.M., Cinatl, J., Eickmann, M., Escriou, N., Grywna, K., Kramme, S., Manuguerra, J.C., Muller, S., Rickerts, V., Sturmer, M., Vieth, S., Klenk, H.D., Osterhaus, A.D., Schmitz, H., and Doerr, H.W. 2003. Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N. Engl. J. Med. 348:1967‐1976.
   Du, H., Disney, M.D., Miller, B.L., and Krauss, T.D. 2003. Hybridization‐based unquenching of DNA hairpins on Au surfaces: prototypical “molecular beacon” biosensors. J. Am. Chem. Soc. 125:4012‐4013.
   Fang, X., Li, J.J., Perlette, J., Tan, W., and Wang, K. 2000. Molecular beacons: Novel fluorescent probes. Anal. Chem. 72:747A‐753A.
   Gilman, M. 2002. Preparation of cytoplasmic RNA from tissue culture cells. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 4.1.1‐4.1.5. John Wiley & Sons, Hoboken, N.J.
   Higuchi, R., Fockler, C., Dollinger, G., and Watson, R. 1993. Kinetic PCR analysis: Real‐time monitoring of DNA amplification reactions. Biotechnology (N Y) 11:1026‐1030.
   Horejsh, D., Martini, F., Poccia, F., Ippolito, G., Di Caro, A., and Capobianchi, M.R. 2005. A molecular beacon, bead‐based assay for the detection of nucleic acids by flow cytometry. Nucleic Acids Res. 33:e13.
   Klein, D. 2002. Quantification using real‐time PCR technology: Applications and limitations. Trends Mol. Med. 8:257‐260.
   Ksiazek, T.G., Erdman, D., Goldsmith, C.S., Zaki, S.R., Peret, T., Emery, S., Tong, S., Urbani, C., Comer, J.A., Lim, W., Rollin, P.E., Dowell, S.F., Ling, A.E., Humphrey, C.D., Shieh, W.J., Guarner, J., Paddock, C.D., Rota, P., Fields, B., DeRisi, J., Yang, J.Y., Cox, N., Hughes, J.M., LeDuc, J.W., Bellini, W.J., and Anderson, L.J. 2003. A novel coronavirus associated with severe acute respiratory syndrome. N. Engl. J. Med. 348:1953‐1966.
   Leone, G., van Schijndel, H., van Gemen, B., Kramer, F.R., and Schoen, C.D. 1998. Molecular beacon probes combined with amplification by NASBA enable homogeneous, real‐time detection of RNA. Nucleic Acids Res. 26:2150‐2155.
   Li, J., Tan, W., Wang, K., Xiao, D., Yang, X., He, X., and Tang, Z. 2001. Ultrasensitive optical DNA biosensor based on surface immobilization of molecular beacon by a bridge structure. Anal. Sci. 17:1149‐1153.
   Liu, X. and Tan, W. 1999. A fiber‐optic evanescent wave DNA biosensor based on novel molecular beacons. Anal. Chem. 71:5054‐5059.
   Liu, X., Farmerie, W., Schuster, S., and Tan, W. 2000. Molecular beacons for DNA biosensors with micrometer to submicrometer dimensions. Anal. Biochem. 283:56‐63.
   Marras, S.A., Kramer, F.R., and Tyagi, S. 1999. Multiplex detection of single‐nucleotide variations using molecular beacons. Genet. Anal. 14:151‐156.
   Matsuo, T. 1998. In situ visualization of messenger RNA for basic fibroblast growth factor in living cells. Biochim. Biophys. Acta 1379:178‐184.
   Mhlanga, M.M. and Malmberg, L. 2001. Using molecular beacons to detect single‐nucleotide polymorphisms with real‐time PCR. Methods 25:463‐471.
   Moore, D. and Dowhan, D. 2002. Purification and concentration of DNA from aqueous solutions. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 2.1.2‐2.1.10. John Wiley & Sons, Hoboken, N.J.
   Ramachandran, A., Flinchbaugh, J., Ayoubi, P., Olah, G.A., and Malayer, J.R. 2004. Target discrimination by surface‐immobilized molecular beacons designed to detect Francisella tularensis. Biosens. Bioelectron. 19:727‐736.
   Sanchez, J.A., Pierce, K.E., Rice, J.E., and Wangh, L.J. 2004. Linear‐after‐the‐exponential (LATE)‐PCR: An advanced method of asymmetric PCR and its uses in quantitative real‐time analysis. Proc. Natl. Acad. Sci. U.S.A. 101:1933‐1938.
   Sokol, D.L., Zhang, X., Lu, P., and Gewirtz, A.M. 1998. Real time detection of DNA. RNA hybridization in living cells. Proc. Natl. Acad. Sci. U.S.A. 95:11538‐11543.
   Tsourkas, A., Behlke, M.A., and Bao, G. 2002. Structure‐function relationships of shared‐stem and conventional molecular beacons. Nucleic Acids Res. 30:4208‐4215.
   Tyagi, S., Bratu, D.P., and Kramer, F.R. 1998. Multicolor molecular beacons for allele discrimination. Nat. Biotechnol. 16:49‐53.
   Tyagi, S. and Kramer, F.R. 1996. Molecular beacons: Probes that fluoresce upon hybridization. Nat. Biotechnol. 14:303‐308.
   Vet, J.A., Majithia, A.R., Marras, S.A., Tyagi, S., Dube, S., Poiesz, B.J., and Kramer, F.R. 1999. Multiplex detection of four pathogenic retroviruses using molecular beacons. Proc. Natl. Acad. Sci. U.S.A. 96:6394‐6399.
   Wang, D., Urisman, A., Liu, Y.T., Springer, M., Ksiazek, T.G., Erdman, D.D., Mardis, E.R., Hickenbotham, M., Magrini, V., Eldred, J., Latreille, J.P., Wilson, R.K., Ganem, D., and DeRisi, J.L. 2003. Viral discovery and sequence recovery using DNA microarrays. PLoS Biol. 1:E2
   Yao, G. and Tan, W. 2004. Molecular‐beacon‐based array for sensitive DNA analysis. Anal. Biochem. 331:216‐223.
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