BeadCons: detection of nucleic acid sequences by flow cytometry.
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.
Horejsh
Douglas
D
National Institute for Infectious Diseases "L. Spallazani"-IRCSS, Rome, Italy.
Martini
Federico
F
Capobianchi
Maria Rosaria
MR
D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D015345:Oligonucleotide Probes; D012680:Sensitivity and Specificity
D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D004247:DNA