Modified method to detect PCR products by 5' nuclease activity and an asymmetric fluorogenic probe set.

PCR products are detected most commonly using postamplification agarose gel electrophoresis and/or DNA hybridization. For reducing time in detection and confirmation, a number of investigators have reported methods to simplify or even eliminate the need to perform postamplification assays. For example, molybodate (2) or some intercalating agents (3) have been used to measure the degree of DNA amplification. However, these protocols are limited because they do not regard PCR product sequence specificity. Other investigators have focused their efforts on adapting the fluorescence resonance energy transfer (FRET) phenomenon to the detection of specific products. Morrison et al. (7) demonstrated that a dsDNA molecule, in which one strand was labeled on the 5′ terminus with fluorescein and the complementary strand was labeled on the 3′ terminus with a quencher, could be used as a tool to detect specific DNA fragments in a postPCR competitive hybridization assay format. Later, the AmpliSensor (Biotronics Technologies, Lowell, MA, USA) protocol (10) eliminated the postPCR competitive hybridization of this original design but required an asymmetric PCR and a subsequent seminested amplification to achieve adequate fluorescent signal. Other improved and enhanced versions of FRET-based PCR protocols have included the TaqMan assay (Applied Biosystems, Foster City, CA, USA) (4,5,6), molecular beacons (9), Amplifluor (Intergen, Purchase, NY, USA) (8), “Scorpions” primers (11) and adjacent hybridization probes (12). In an effort to reduce the cost associated with the production of dual-labeled fluorescent probes for such assays, an asymmetric fluorogenic probe set was designed and tested for its applicability to the rapid endpoint detection of PCR products. The procedure described in this report is outlined in Figure 1. A long 5′ fluorescein-labeled (reporter) oligonucleotide probe and a short 3′ DABCYL (4-[4′-dimethylamino phenylazo] benzoic acid)-labeled (quencher) oligonucleotide were used in this modified system. Because these two oligonucleotides contained complementary sequences, they are able to form an asymmetrical double-stranded conformation such that the 5′ fluorescein-labeled probe is quenched by the nearby quencher oligonucleotide. However, since the two probes share only a short complementary region, the annealing temperature of the fluorescein-labeled probe is quite low. During the normal annealing/primer extension step of PCR, we hypothesized that most of the reporter probe molecules are available to hybridize with the target PCR products, after which they are digested by the 5′ nuclease activity of Taq DNA polymerase. Although the number of short quencher oligonucleotides (competitors) in the reaction solution is quite high, the elevated temperatures of denaturation and extension should hinder the annealing between the reporter and quencher probes. After the amplification is complete, the residual reporter probe will re-anneal to the short quencher probe at room temperature. An overall increase of fluorescence at the end of the assay should signal detection of the specific target DNA product, while the intensity of the signal should be roughly equivalent to the quantity of amplified DNA. In our model system, the Listeria monocytogenes listeriolysin O (hly) gene was used as the PCR amplification target. All the amplification primers, reporter and quencher probes were synthesized by Integrated DNA Technologies (Coralville, IA, USA). The PCR primers (forward primer: 5′-AGGATGCATCTGCATTCAA-3′ and reverse primer: 5′-GGATATCTGCATTATTTTGATT-3′) were designed based on DNA sequence data from GenBank (accession nos. U25443, U25446, U25449 and U25452). A 273-bp hly gene fragment was amplified directly from 1 μL overnight culture of L. monocytogenes ATCC 19115 and cloned into the pT7Blue-3 cloning vector (Novagen, Madison, WI, USA) as a positive control DNA template. The fluoresceinBenchmarks