New qPCR technologies involving molecular beacons were described by Fred Kramer, Ph.D., professor at New Jersey Medical School. Like the faithful lighthouses that send out signals to ships at sea, molecular beacons provide biological signals useful in applications that range from rapid pathogen identification to genetic screening.
“We have been developing ways to rapidly identify potentially lethal sepsis-causing bacteria. Identification in a clinical setting often takes several days, during which time a patient is treated with a broad-spectrum antibiotic. This strategy occasionally fails and contributes to the development of antibiotic-resistant strains. With a molecular-beacons approach in a qPCR setting, we can identify pathogens in one reaction in only one or two hours.”
Molecular beacons are single-stranded oligonucleotide hybridization probes with a unique architecture. They form a stem-and-loop structure in which the loop contains a probe sequence complementary to the target. One terminus of the stem contains a fluorophore and the other a quencher. Like a molecular switch, molecular beacons light up only when the probe hybridizes to its target.
According to Dr. Kramer, molecular beacons can be designed to be either “finicky” or “sloppy”. “Finicky probes are shorter (~18–26 nucleotides) and form hybrids with perfect complementarity. These are useful for genetic screening, detection of SNPs, and pharmacogenetic applications. Sloppy probes, however, are longer (~40 nucleotides) and hybridize to a broad range of species we want to identify.”
This feature helps identify pathogens in a clinical sample. “We use a sloppy probe set with four differently colored fluorophores in a single gene amplification reaction,” he said. “The molecular beacons hybridize at a relatively low temperature. As the temperature is slowly raised, each of the four probe-target hybrids melts apart and signal is lost. The resulting set of four melting temperatures creates a specific signature that identifies which of potentially hundreds of species is present in the sample.”
Dr. Kramer has licensed the molecular-beacons technology for an assortment of clinical tests that range from HIV-1 identification to tuberculosis screening. While his current research is still at the development stage, he is optimistic that advanced molecular-beacon designs will enable new clinical diagnostic applications.