There are also a number of 3-probe combinations that should be suitable for use in a multiplex assay, for example, FAM and CY5 with HEX, CR 6G, CY3, or TAMRA (see Table ). It is also possible that a 4-probe combination such as FAM, HEX, TAMRA, and CY5 could be resolved.
Molecular beacons, like other oligonucleotides, will readily adhere to untreated plastic. Therefore, it is important to use microplates which have been surface treated to minimize binding. We have found that plates treated for cell culture exhibit less binding than do untreated plates.
Molecular beacons, like other analytical tools, have potential pitfalls. Depending on the temperature, they will react with targets containing one or more nucleotide mismatches.24 Temperature control is especially important for discrimination between perfectly complementary targets and targets containing a single-nucleotide mismatch.3,4
Tyagi et al. state that fluorescence increases as much as 900-fold when probes bind to their target2. In practice, the ratios are typically much lower. A high ratio is obviously desirable because it affords high sensitivity. A low ratio can be due to a number of factors, including contamination with free fluorophore and contamination with oligonucleotides that are missing the quencher. At least three commercial suppliers guarantee a minimum ratio of 25 (Research Genetics; Synthegen, Houston, Texas; and Pacific Oligos, Toowong, Australia).
The reaction of molecular beacons with their targets can be measured in the SPECTRAmax GEMINI micr