Folding of amplicons into any kind of secondary structures is usually avoided in PCR. This is because secondary structures in the template strand can impede the progression of DNA polymerases.34–36
Further, it is well established that the secondary structures negatively affect the hybridization properties of oligonucleotides.37,38
Snake is the first DNA detection technology that is deliberately based on PCR amplicon folding. The previous study13
showed that up to a certain level of stability these secondary structures do not interfere with the amplification process but effectively catalyze the FRET probe cleavage. However, the ideal range in thermodynamic stability of the folded PCR amplicons, which provides all detection benefits at no cost to the PCR efficiency, is relatively narrow. The goal of the present study was to identify the reaction conditions and approaches that would enable the expansion of this thermodynamic range, simplify the system design and further improve the assay performance.
One of the identified methods is the use of template-efficient duplex-destabilizing modifications 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. The duplex-destabilizing modifications in 5′-flaps are especially effective when applied in combination with asymmetric three-step PCR. This is because the introduction of the PCR extension step helps to overcome yet another problem associated with the sense amplicon folding ().
Perhaps the most valuable discovery of the present study is the use of base-modified duplex-stabilizing dNTPs in Snake PCR. This approach made possible the application of very short 6-8-mer 5′-flap sequences in the Snake primers. According to Lyamichev and coworkers,39
the cleaved duplex (antisense amplicon folding) may not be shorter than ~9-mer to support the 5′-nuclease cleavage. For the Snake technology, this means that use of the very short flap sequences (<9-mers) can be an effective solution to the problem of the antisense amplicon cleavage (). These estimates of the minimum duplex length may not be accurate, but the results of the present study are in good agreement with them. The Snake assays employing the shortest 6-8-mer flaps in the forward primers were the best real-time systems used so far for detection of the β2-microglobulin target (compare data in and
An important feature of the Snake technology is its ability to use very short FRET probes. In the present study, for example, a 9-mer FRET probe performed in Snake better than a homologous 22-mer probe in TaqMan. The 9-mer probe's performance in Snake looks especially impressive in light of the fact that the Tm
of this oligonucleotide (Tm
46°C, ) was as much as 10° lower than the PCR detection temperature of 56°C (annealing stage). Actually, the trend in the probe length reduction—originally from 22-mer to 15-mer13
and then, in the present study, to 9-11-mer sequences—is rapidly approaching the 6-8-mers boundary of a universal library.40
Establishment of a complete FRET probe inventory is projected to have stimulating and long-lasting consequences for nucleic acids research mainly due to a considerable, up to ~10-fold reduction in detection cost. Future research will show whether or not this milestone can be achieved using the Snake technology. Note, however, that powerful potentials are still left unexplored. The present 9-mer-probe-length record has been achieved without applying any duplex-stabilizing modifications in the probe structure. The toolbox of such modifications is actually rich and well represented.25–32
The Taq DNA polymerase used in the study does not have 3′→5′ exonuclease proofreading activity. Consequently, more than half of the PCR products may incorporate an extra nucleotide residue, usually adenosine, at the 3′ end.41
This is an undesired process in Snake because it makes a fraction of the sense amplicons incorporate not 3′-mono but 3′-dinucleotide mismatched termini and this does not support the 5′-nuclease cleavage. Further, appearance of the mismatched nucleotide at the 3′-end of the truncated sense amplicon (stages D and E, ) would prevent this amplicon from repairing in the pathway E→F. The efficiency of the extra nucleotide incorporation in Snake remains unclear. However, due to the superior assay signal and other evidence, it is unlikely to exceed ~20%–30%.
Even with the progress achieved so far, the Snake system is far from its complete optimization. Snake is an enabling technology. It can be a key component or work in combination with other methods in the development of new and advanced molecular tools. In addition to demonstrating the validity of the hypothesis and concepts, the use of the same β2-microglobulin target throughout the study enables accurate comparison of various methods and also keeps the R&D budget low. The next logical step is the validation of the Snake technology on numerous genomic DNA targets. This and other interesting Snake-related projects are presently underway at Perpetual Genomics and the results will be published very soon.