Real Time PCR Using Molecular Beacons : A New Tool to Identify Point Mutations and to Analyze Gene Expression in Mycobacterium tuberculosis

Abstract

Molecular beacons are a novel family of hybridization probes, which emit fluorescence upon interaction with their target. They are hairpin-shaped oligonucleotides with a central part complementary to the target, flanked by two 5 6 base pair (bp) inverted repeats, which can form a stable stem. A fluorescent moiety is covalently linked to the 5' end of the molecule, whereas the quenching moiety, 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL), is covalently linked to the 3' end. The stem keeps the two moieties in close proximity to each other, causing the fluorescence of the fluorophore to be quenched by energy transfer. When molecular beacons bind to their target, they undergo a conformational change that results in the restoration of fluorescence of the internally quenched fluorophore (1) (Fig. 1). Molecular beacons are extremely specific, and can clearly discriminate between targets differing only by a single nucleotide (2,3). When present in a PCR reaction where their target is the amplification product, molecular beacons can form a stable hybrid with the amplicon during the annealing step. The intensity of fluorescence at the annealing step in each amplification cycle is a direct measure of amplicon concentration (2,4) (Fig. 2). Another interesting feature of molecular beacons is that they can be coupled to a variety of differently colored fluorophores. This allows multiplex PCR reactions where different DNA fragments can be amplified and detected simultaneously in the same tube (2,3). Fig. 1. Operation of molecular beacons. On their own, these molecules are nonfluorescent, because the stem hybrid keeps the fluorophore (◯) close to the quencher (•). When the probe sequence in the loop hybridizes to its target, forming a rigid double helix, a conformational reorganization occurs that separates the quencher from the fluorophore, restoring fluorescence (1). Fig. 2. Real time measurement of amplicon synthesis during PCR using molecular beacons. (A) Four PCR reactions were initiated with a different number of template molecules (indicated). The concentration of amplicons present after each cycle of amplification was determined by measuring fluorescence during the last few seconds of the annealing step. (B) Inverted relationship between the threshold cycle (the cycle at which the fluorescent signal becomes detectable above the background) and the logarithm of the initial number of template molecules. In this example, the target is M. tuberculosis H37Rv chromosomal DNA. The primers-molecular beacon set used in the reaction was specific for sigA (reprinted from ref. 4).