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BeadCons: Detection of Nucleic Acid Sequences by Flow Cytometry

Douglas Horejsh1,  Federico Martini1,  Maria Rosaria Capobianchi1

1National Institute for Infectious Diseases “L. Spallazani”‐IRCSS, Rome, Italy, 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

  • Unit Introduction
  • Basic Protocol: 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: Design of Molecular Beacons for use in a Beadcons Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

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

 Materials
  • 10 µM biotinylated molecular beacon (see recipe; see Support Protocol 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

 Additional Materials (also see Basic Protocol)
  • 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

 Additional Materials (also see Basic Protocol or Alternate Protocol 1)
  • 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 H2O (Gilman, 2002)
  • 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 MgCl2
  • Oligonucleotide PCR primers (target sequence–specific)
  • BeadCons recognizing (–) strands of sequences of interest (see Basic Protocol or Alternate Protocol 1)
  • Microcentrifuge tubes or PCR tubes
  • Thermal cycler
  • Additional reagents and equipment for DNA extraction (Moore and Dowhan, 2002)
  • 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. (2005) for further information.

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

 Additional Materials (also see Basic Protocol and Alternate Protocol 1)
  • BeadCons (each target identified by a fluorescent dye and bead size combination; see Basic Protocol or Alternate Protocol 1)
  • 1 µM template nucleic acids, test samples and controls (see Alternate Protocol 2 for preparation)

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

 Materials
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Figures

  • Figure 13.5.1
    Steps in performing a BeadCons assay.

    View Image
  • Figure 13.5.2
    Structure of a biotinylated molecular beacon used in the construction of a BeadCon. The molecular beacon sequence for severe acute respiratory syndrome virus nucleocapsid protein (SARS-N) is shown with the stem region shaded and the loop region outlined. The 5¢ fluorochrome in this example is 6-carboxyfluorescein (6-FAM), paired with Black Hole Quencher-1 (BHQ-1) as the 3¢ quencher. Biotinylation of a thymidine residue within the 3¢ stem, close to the quencher molecule, allows streptavidin binding without interfering with the molecular beacon function.

    View Image
  • Figure 13.5.3
    Structure and analysis of a BeadCon constructed with direct conjugation. Signal-to-noise ratio (SNR) is determined by comparing the BeadCon mean fluorescence intensity (MFI) after hybridization with a positive control oligo with that of a negative control oligo.

    View Image
  • Figure 13.5.4
    Structure and analysis of a BeadCon constructed with streptavidin-bridged conjugation. The signal-to-noise ratio (SNR) is determined by comparing the BeadCon mean fluorescence intensity (MFI) after hybridization with a positive control oligo with that of a negative control oligo. The SNR observed after using bridged BeadCons typically approaches a two-fold increase in assay range over the SNR using BeadCons constructed with direct conjugation; therefore, the bridged construction method is recommended.

    View Image
  • Figure 13.5.5
    Dot plots demonstrating positive, qualitative detection of an asymmetric PCR product derived from a clinical sample. A nucleic acid sequence–based amplification (NASBA) preparation was used as a template in a reverse transcription (RT) reaction to produce cDNA as described in Alternate Protocol 2. PCR reactions were carried out with no primers (data not shown), symmetrically with 50 pmol each primer (1), or asymmetrically with 1 pmol (+) primer and 50 pmol (–) primer (2). The dotted line indicates the negative control cutoff.

    View Image
  • Figure 13.5.6
    Readout of nucleic acids using the BeadCons assay. (A) Histogram plot after flow cytometric analysis of BeadCons in a single target format. The BeadCons exhibit a positive fluorescence shift (shaded peak) after addition of the corresponding oligonucleotide sequence. (B) Qualitative detection of sequences in multiplex. A BeadCons array was generated using two bead sizes, 3 µm and 7 µm, and two fluorescence markers, 6-FAM and Cy5. Combinations are indicated by the numbers 1, 2, 3, and 4. The addition of target DNA sequences, DNA 2 and DNA 3, diluted in a complex DNA mixture (5% specific target and 95% non specific target oligos) allow the specific identification of the target compared to negative controls. Arrows denote positive fluorescence shifts in the presence of target DNA 2 and 3. In effect, internal negative controls are shown for each BeadCon, as each test was done using a complex DNA mixture.

    View Image

Literature Cited

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