Use of Base Modifications in Primers and Amplicons to Improve Nucleic Acids Detection in the Real-Time Snake Polymerase Chain Reaction

Abstract

The addition of relatively short flap sequence at the 5 ¢ -end of one of the polymerase chain reaction (PCR) primers considerably improves performance of real-time assays based on 5 ¢ -nuclease activity. This new technology, called Snake, was shown to supersede the conventional methods like TaqMan, Molecular Beacons, and Scorpions in the signal productivity and discrimination of target polymorphic variations as small as single nucleotides. The present article describes a number of reaction conditions and methods that allow further improvement of the assay performance. One of the identified approaches is the use of duplex-destabilizing modifications such as deoxyinosine and deoxyuridine in the design of the Snake primers. This approach was shown to solve the most serious problem associated with the antisense amplicon folding and cleavage. As a result, the method permits the use of relatively long-in this study-14-mer flap sequences. Investigation also revealed that only the 5 ¢ -segment of the flap requires the deoxyinosine/ deoxyuridine destabilization, whereas the 3 ¢ -segment is preferably left unmodified or even stabilized using 2-amino deoxyadenosine d(2-amA) and 5-propynyl deoxyuridine d(5-PrU) modifications. The basemodification technique is especially effective when applied in combination with asymmetric three-step PCR. The most valuable discovery of the present study is the effective application of modified deoxynucleoside 5 ¢ -triphosphates d(2-amA)TP and d(5-PrU)TP in Snake PCR. This method made possible the use of very short 6-8-mer 5 ¢ -flap sequences in Snake primers.

Use of extremely short Förster resonance energy transfer probes in real-time polymerase chain reaction

“…However, this is not an option for the Angler assay. Folding of the PCR amplicons slows down the primer hybridization (25,26), and this was shown to negatively affect PCR even in a less complex system like Snake (18,27). For this reason, the PCR profile used in this study comprised three steps.…”

“…Reflecting the system’s complexity, the Angler assay detection is delayed by a few cycles (Figure 4B), but most importantly it upholds the linear dependence between the threshold values and the logarithm of the target loads (R 2 > 0.99). The Angler predecessor, the Snake system was found to positively respond to an excess of the reverse primer (18,27) in asymmetric PCR (30). The Angler assay apparently follows the same trend.…”

A “turn on/off” scorpion biosensor targeting point mutation of SMN genes for diagnosis of spinal muscular atrophy

“…1 Fluorometric detection of PCR products is a popular detection mode which simplifies the readout procedure and can use probes for diagnosis. 2 Fluorescence resonance energy transfer (FRET) is a process in which the energy from an excited fluorophore is transferred to an acceptor moiety at distances of up to 7-10 nm. 3,4 At present, there are some probe-based reporter systems, such as TaqMan, 5,6 molecular beacons [7][8][9] and the scorpions.…”