Use of extremely short Förster resonance energy transfer probes in real-time polymerase chain reaction.
Bottom Line: The method is based on the production of PCR amplicons, which fold into dumbbell-like secondary structures carrying a specially designed 'probe-luring' sequence at their 5' ends.The unparalleled cost-effectiveness of the inventory approach is discussed.Despite the short length of the probes, this new method, named Angler real-time PCR, remains highly sequence specific, and the results of the study indicate that it can be effectively used for quantitative PCR and the detection of polymorphic variations.
Affiliation: Perpetual Genomics, Woodinville, WA 98077, USA.
Described in the article is a new approach for the sequence-specific detection of nucleic acids in real-time polymerase chain reaction (PCR) using fluorescently labeled oligonucleotide probes. The method is based on the production of PCR amplicons, which fold into dumbbell-like secondary structures carrying a specially designed 'probe-luring' sequence at their 5' ends. Hybridization of this sequence to a complementary 'anchoring' tail introduced at the 3' end of a fluorescent probe enables the probe to bind to its target during PCR, and the subsequent probe cleavage results in the florescence signal. As it has been shown in the study, this amplicon-endorsed and guided formation of the probe-target duplex allows the use of extremely short oligonucleotide probes, up to tetranucleotides in length. In particular, the short length of the fluorescent probes makes possible the development of a 'universal' probe inventory that is relatively small in size but represents all possible sequence variations. The unparalleled cost-effectiveness of the inventory approach is discussed. Despite the short length of the probes, this new method, named Angler real-time PCR, remains highly sequence specific, and the results of the study indicate that it can be effectively used for quantitative PCR and the detection of polymorphic variations.
Mentions: All oligonucleotide components in Angler PCR, the primers and probes, carry special tail sequences that are critical for the assay performance. Figure 2 illustrates how the Angler system functions. Similar to the Snake technology (18), the forward PCR primer contains a 5′-flap sequence (light blue) called the ‘cleavage-enhancing’ flap, which is complementary, save for the 5′-terminal base, to the product of this primer extension, downstream from the original primer sequence. Extension of this forward primer in stage I results in the synthesis of an antisense strand, providing a double-stranded amplicon II. After strand separation (95°C), a reverse primer hybridizes to the antisense strand and DNA polymerase extends the complex (stage III), resulting in yet another double-stranded amplicon IV. The extension of the reverse primer generates a replica of the 5′-flap sequence of the forward primer at the 3′ end of the sense strand (dark blue). The reverse primer also incorporates a 5′-flap sequence. This flap sequence comprises two segments. The first segment (red), named ‘probe-directing’, is designed to fold the sense amplicon into a stem-loop structure. The second segment (green), named ‘probe-luring’, is an artificial nucleotide sequence that is designed to be complementary to a corresponding anchoring sequence (also green) conjugated to the 3′ end of a FRET probe. After strand separation of the double-stranded amplicon IV, the sense amplicon folds into a dumbbell-like secondary structure V. The short probe (yellow), except its anchoring 3′ tail, is complementary to the target sequence of the sense amplicon located between the cleavage-enhancing and probe-directing duplexes. Hybridization of the probe to the folded sense amplicon results in the formation of a three-way DNA junction VI, or Y-structure for short. The flap and tail sequences in the primers and probe are selected in length and base composition to make all three key duplexes in the structure VI relatively stable at the PCR detection temperature.Figure 2.