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gene_expression

Molecular beacon probes

Improved signal-to-noise ratio with FRET-based hybridization probes

Molecular Beacons are dual-labeled probes that form a quenched, stem-loop structure in native state and fluoresce upon hybridization to the target nucleotide sequence. Applications include real-time and endpoint PCR, SNP detection, and multiplex amplification.

  • Detect single mismatch from target with hybridization probes
  • Perform post-PCR melt curve analysis, if needed, since probes are not hydrolyzed for signal generation
  • Choose from a wide variety of reporter and quencher combinations
  • Start experiments sooner: standard probes ship in 5–7 business days

Ordering

Prices listed include probe sequence (up to 45 bases in length), reporter, quencher, and HPLC purification. Turnaround time is 5–7 business days for standard molecular beacon probes.

To request molecular beacon probes with <18 or >45 bases, please contact our Technical and Customer Support.

Molecular beacon probes

Hairpin-shaped, fluorescent, dual-labeled nucleic acid probes that derive enhanced binding specificity from conformational changes during hybridization.

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Molecular beacon probess are qPCR probes that contain a 5′ dye and 3′ quencher, and are designed to form a stem-loop (hairpin) structure. The loop contains sequences that hybridize to the target DNA and is flanked by complementary arms (typically 5–7 bp long) that form the stem, when the arms hybridize to each other. Because perfectly matched hybridization between the probe and target sequences is thermodynamically favored over the hairpin structure, and because the hairpin structure is thermodynamically favored over hybridization to a non-specific target sequence, molecular beacon probes are highly specific. The following steps are repeated for each PCR cycle and allow detection of specific products.

Step 1: During the annealing step, hybridization of molecular beacon probes to target sequences separate the fluorescent dye (D) and quencher (Q), resulting in fluorescence that is detected by the real-time PCR instrument. Probes that do not hybridize to target DNA sequences will reform the hairpin structure and will not fluoresce.

Step 2: The polymerase extends from the primers and begins DNA synthesis.

Step 3: When the polymerase reaches the molecular beacon, the probe is displaced without being degraded. Therefore, molecular beacons can participate in multiplex rounds of annealing.

Step 4: The polymerase continues extension of the primers to complete synthesis of the DNA strand.

Recommended reading

  1. Tyagi S, Kramer FR. (1996) Molecular beacons: probes that fluoresce upon hybridization. Nat Biotechnol, 14:303–308.
  2. Bonnet G, Tyagi S, et al. (1999) Thermodynamic basis of the enhanced specificity of structured DNA probes. Proc Natl Acad Sci USA, 96:6171–6176.

Frequently asked questions

OligoAnalyzer Tool

Identifies oligonucleotide properties, including melting temperature, hairpins, dimers, and mismatches

UNAFold Tool

Performs secondary structure analysis