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Assay Drug Dev Technol. Feb 2011; 9(1): 58–68.
PMCID: PMC3033208
Use of Base Modifications in Primers and Amplicons to Improve Nucleic Acids Detection in the Real-Time Snake Polymerase Chain Reaction
Igor V. Kutyavincorresponding author
Perpetual Genomics, Woodinville, Washington.
corresponding authorCorresponding author.
Address correspondence to: Igor V. Kutyavin, Ph.D., Perpetual Genomics, 18943 203rd Ave. NE, Woodinville, WA 98077. E-mail:perpetualgenomics/at/comcast.net
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 base-modification 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.
Polymerase chain reaction (PCR) is the most commonly used DNA amplification technique, and the PCR-based methods are capable of detecting as little as a single copy of DNA or RNA. Fluorimetric detection of PCR products has simplified readout and made possible real-time techniques that allow amplification to be monitored continuously.1,2 The most advanced and target-specific methods are based on probe detection. Fluorescent probes are oligonucleotides designed to bind exclusively to a target amplicon and they are usually synthesized with both a reporter fluorescent dye and a quencher dye that are in Förster resonance energy transfer (FRET) interaction.3 When FRET occurs, emission of the reporter dye is extinguished by the quencher. Disruption of FRET by dye separation results in a fluorescent signal. This effect is widely used in probe designs for nucleic acid detection.4,5
The best strategy to abolish FRET is based on cleavage of the oligonucleotide probe upon its binding to a target nucleic acid. When cleavage takes place anywhere between the conjugated dyes, the result is a complete and irreversible disruption of FRET. Although a number of ways to achieve the probe cleavage have been explored,68 the TaqMan technology was the first developed9 and it remains widely used for real-time nucleic acid detection in PCR.10 The method utilizes the 5′- nuclease activity of Thermus aquaticus (Taq) DNA polymerase. A dual-labeled FRET probe is designed to anneal to a target sequence located between two PCR primer binding sites. During strand elongation, Taq polymerase cleaves the probe that is hybridized down stream from a primer site, releasing the reporter dye from the quencher. Unlike in the hybridization-triggered assays, for example, Molecular Beacons11 and Scorpion primers,12 the TaqMan probe signal generated at a given PCR cycle is the sum of signals generated at that particular cycle plus all previous ones. However, the elevated fluorescence background of the TaqMan probes overshadows the signal advantage. Attempts to compare the performances of various probe-based technologies in real-time PCR are extremely rare, but an example can be found in Ref.13 Figure 1 illustrates the system designs and the mechanism of the signal generation in the most commonly used technologies for real-time PCR.
Fig. 1.
Fig. 1.
The diagrams show the systems' design, the key reaction stages, and the mechanism of the fluorescence signal generation in the probe-based technologies most commonly used in real-time PCR. TaqMan (A) is a rare example of the methods wherein FRET is abolished (more ...)
A new technology for real-time PCR detection of nucleic acids was recently described.13 Similar to TaqMan, this new method, named Snake, also utilizes the 5′-nuclease activity of Taq DNA polymerase. However, the mechanism of the probe cleavage in Snake is different. In this assay, PCR amplicons fold into stem-loop secondary structures. Hybridization of FRET probes to one of these structures leads to the formation of optimal substrates for the 5′-nuclease of Taq. The stem-loop structures in the Snake amplicons are introduced by the unique design of one of the PCR primers, which carries a special 5′-flap sequence. The Snake system favors the use of relatively short FRET probes with improved fluorescence background, and it was shown to supersede all conventional assays in the signal productivity and detection of sequence variations as small as single nucleotide polymorphism (SNP). However, this seemingly ideal technology for real-time PCR has its own Achilles heel. Length and base composition of the 5′-flap sequences in Snake primers determines stability of secondary structures in the folded PCR amplicons. Unstable structures have little or no positive effect on the assay, whereas overstabilization leads to reduction in PCR yield. Nonetheless, for any specific PCR condition there is always a range in thermodynamic stability of the folded amplicons within which there is essentially no negative effect on PCR but there is considerable benefit to the 5′-nuclease signal productivity. Design of the 5′-flap primers to enter this range is difficult in the absence of specialized software. The present study aims to overcome this inconvenience. In particular, it shows that certain nucleotide base modifications and changes in PCR protocol help to remove the stringencies in the Snake primer design, allowing effective use of both very long and very short 5′-flap sequences.
PCR Components
JumpStart™ Taq DNA polymerase, an antibody-inactivated hot start enzyme, and regular dNTPs were purchased from Sigma-Aldrich. Base-modified dNTPs were obtained from TriLink Biotechnologies. Primers and FRET probes for TaqMan and Snake assays were prepared by Cepheid using reagents from Glen Research. The sequences of oligonucleotides used in this study are shown in Figure 2. FAM in FRET probes is 6-fluorescein and BHQ1 is a Black Hole Quencher™ from Biosearch Technologies. The oligonucleotide 2 μM stock solutions were prepared and stored refrigerated in 5 mM sodium cacodylate.
Fig. 2.
Fig. 2.
The scheme shows the sequences of FRET probes and PCR primers used in the TaqMan and Snake assays of the present study. Wherever it is possible, the primers and probes are aligned with a detected sequence of 96-mer target oligodeoxyribonucleotide. The (more ...)
Physical Measurements and Calculations
Oligonucleotide extinction coefficients were calculated using CalcExt2.8 software (Cepheid). Melting temperatures (Tms) of unmodified primers and probes were calculated using the nearest-neighbor approach,15,16 for perfect-match duplexes at 200 nM with adjustment for the PCR buffer used in this study. The effect of dyes on duplex stability was disregarded in the calculations.
Each real-time curve shown here represents an average of at least 3–5 independent experiments. The fluorescence curve threshold values (Ct) were determined using semi-log fluorescence plotted versus cycle number. In all cases reported here, the threshold values represent the cycle number at which the linear trend of the semi-log fluorescence curve reached 0.5. Microsoft Excel was used to establish the linear trend within 4–5 data points (cycles) with values of fluorescence logarithm >0.2. The reported Ct values were averaged based on 3–5 experiments and rounded to an integer. In the case of more accurate Ct measurements, confidence intervals were calculated and reported for 95% confidence level based on 5–8 independent experiments. A relative change in PCR yield ΔY = 100 × (1  x)% was calculated based on the equation 2N = (1 + x)n, wherein n and N are, respectively, the Ct values of the discussed and referenced reactions.
DNA Target
A 96-mer synthetic DNA oligonucleotide (Sigma-Genosys) containing a portion of the β2-microglobulin gene (GenBank accession no. NM004048) was used as a target for PCR. The sequence shown in Figure 2 is a fragment of the target between the 5′-CG…and…CTA-3′ ends. To prevent adsorption of the target DNA to plastic tubes at subnanomolar concentrations, it was diluted in 100 nM (dA)18 conjugated at the 3′-end with propane diol.
Real-Time PCR
PCRs were conducted in 25 μL volumes and contained 104 molecules of the target DNA. The standard symmetric PCR was originally optimized for the TaqMan assay, and the reactions included the following: forward and reverse PCR primers at 200 nM concentration each; a FRET probe—200 nM; dNTPs—200 μM each; JumpStart DNA polymerase (Sigma)—0.04 U/μL in 50 mM KCl and 20 mM Tris-HCl (pH 8.0). Magnesium chloride was applied at 2 mM concentration in experiments with the TaqMan and 14-mer-flap Snake primers Pr3a-i (Fig. 2). In the cases of short 6-8-mer flap sequences (Pr4-6, Fig. 2) the MgCl2 concentration was elevated to 5 mM. The asymmetric PCR was different from the symmetric protocol in the primer concentrations (reverse Pr2 vs. forward Snake primer, 400:100 nM) and the amounts of JumpStart DNA polymerase used (0.2 U/μL). In two-step PCR, the reaction mixtures were incubated at 95°C for 2 min to activate the antibody-blocked enzyme followed by 50 PCR cycles consisting of 10 s at 95°C for denaturation and 45 s at a designated annealing temperature X of 56°C or 64°C, that is, (95°2′)→(95°10′′→X°45′′)50. The three-step PCR profile incorporated an additional extension step at 72°C for 15 s, that is, (95°2′)→(95°10′′→X°45′′→72°15′′)50. These differences between the PCR protocols used are discussed throughout the text and also outlined in Table 1. In certain experiments a base-modified dNTP analog such as 2-amino deoxyadenosine 5′-triphosphate [d(2-amA)TP] or 5-propynyl deoxyuridine 5′-triphosphate [d(5-PrU)TP] was completely substituted for the corresponding natural nucleotides, dATP or dTTP. All real-time PCR experiments were performed on SmartCycler (Cepheid). Background fluorescence was subtracted using instrument software and the data (fluorescence vs. PCR cycle) were transferred to an Excel format (Microsoft Office) for further processing. Fluorescence was measured during the annealing stage. The 25 μL reaction volume used in this study is required by SmartCycler. Other real-time instruments allow a volume reduction of up to 5 μL with no effect on the detection.
Table 1.
Table 1.
Effect of Nucleotide Modifications in a 14-Mer 5′-Flap Sequence of Forward Snake Primer on Threshold Values (Ct) of Fluorescence Curves During Real-Time Polymerase Chain Reaction
Use of Deoxyuridine and Deoxyinosine Modifications for Selective Destabilization of Antisense Amplicon Folding
The length of the flap sequences in Snake primers defines the length of the duplex fragments in the folded amplicons. Therefore, elongation of the flap sequences is an effective way to promote amplicon folding and, in particular, folding of the catalytic sense amplicon, which controls the signal productivity in Snake. On the other hand, overstabilization of these stem-loop structures—especially the antisense one—negatively affects PCR yield and may result in unacceptable delay in the signal appearance.13 For example, compared to the standard TaqMan assay, real-time curves in the experiment with 14-mer flap Snake primer Pr3a were almost 4 cycles late in detecting the target DNA (Table 1, ΔCt = + 3.6). Both amplicons, sense and antisense, can slow down PCR but through different mechanisms. Figure 3 illustrates negative consequences of the antisense amplicon folding that is initiated in PCR by cleavage of the 5′-flap sequence in stages B and C. The antisense amplicon is not the one that catalyzes the FRET probe cleavage. Its main purpose is to replicate during PCR. Folding of the antisense amplicon is absolutely unnecessary in Snake.
Fig. 3.
Fig. 3.
Illustrated are the reaction pathways of an antisense Snake amplicon that can have a negative effect on PCR efficiency. Because of the flap sequence, the Snake amplicon folds into a stem-loop secondary structure in stage A. During the extension of a reverse (more ...)
Perhaps the most straightforward way to destabilize the antisense amplicon secondary structure is to use nucleotide modifications in the design of the 5′-flap sequences. However, only a very limited number of the duplex-destabilizing modifications can effectively replicate in PCR. Deoxyuridine (dU) and deoxyinosine (dI) are rare examples of the nucleoside analogs that are not only effective in duplex destabilization,17,18 but are also template efficient and well tolerated by DNA polymerases during PCR.19 For example, dU triphosphate (dUTP) is commonly used in PCR to prevent contamination carryovers from sample to sample,20 whereas dITP and its analogs are effective in amplifying exceptionally G-rich sequences.21 As anticipated, the corresponding modification of all guanosines and thymidines within the 14-mer flap sequence of primer Pr3a (Fig. 2) led to an improvement in the PCR efficiency, reducing the delay in Ct values from +3.6 to +0.9 cycles (compare Pr3a vs. Pr3b, symmetric two-step PCR, Table 1).
Asymmetry in Hybridization Properties Between the 5′ and 3′ Segments of the Flap Sequences: Optimal Snake Primer Design
Actually, the primer flap sequence does not need to be completely destabilized. Cleavage of the folded antisense amplicon takes place primarily at the 5′-end (stage C, Fig. 3). Respectively, the 5′-segment of the flap requires destabilization, whereas the corresponding 3′-segment may be left unmodified. Destabilization of the 3′-segment may have a negative effect on PCR by preventing the sense amplicon replication through the active hybridization process A→F→G→E shown in Figure 4. The weak base pairing of dI-dC and dU-dA does not support the strand displacement of the natural duplexes in the F→G direction (Fig. 4), and the F↔G equilibrium is shifted toward the stage F, thus limiting the reaction to go predominantly through the passive and relatively time-consuming A→E hybridization pathway. According to our previous study,13 this can sensitively slow down the DNA replication. For example, in the case of the 14-mer primer Pr3a, the problem associated with the sense amplicon folding accounted for as much as half of the reduction in PCR yield.
Fig. 4.
Fig. 4.
The scheme shows reaction pathways of a sense Snake amplicon during PCR. Folding of this particular amplicon catalyzes the FRET probe cleavage in stages A→B→C→D, resulting in an exceptionally strong fluorescence signal that is (more ...)
The above logic crystallizes into the following rule. For the best performance, the 5′-segment of the flap sequence has to be destabilized, whereas the corresponding 3′-segment has to be stabilized or, at least, left natural. Analysis of the data shown in Table 1 (two-step symmetric PCR) supports this rule. For example, the forward primer Pr3c, having only the 5′-segment destabilized, was more effective in real-time PCR (ΔCt = +0.3) than its counterpart primer Pr3d (ΔCt = +1.8) or the completely modified primer Pr3b (ΔCt = +0.9). Interestingly, the same trend was observed in the case of the flaps modified with duplex-stabilizing modifications 2-amino deoxyadenosine (2-amA) and 5-propynyl-deoxyuridine (5-PrU) (compare the data for primers Pr3e-g in Table 1). Moreover, the hybrid primer Pr3h, wherein the 5′-flap segment had been destabilized while the 3′-segment stabilized, was actually one of the best performers (ΔCt = +0.3). Its counterpart, the primer Pr3i with a reverse pattern in the flap hybridization disequilibrium was one of the worst performers in Snake (ΔCt = +3.5).
Because of the risk of overstabilizing the antisense amplicon folding, incorporation of duplex-stabilizing 2-amA and 5-PrU modifications throughout the flap sequence should be avoided. For example, the threshold values obtained for primers Pr3e-g are generally higher than those for primers Pr3b-d (Table 1). Nevertheless, these seemingly worse primers, Pr3e-g, provided detection earlier than the unmodified 14-mer flap primer Pr3a (ΔCt = +3.6). There is an explanation for this. Stabilization of the flap does not add much to the cleavage efficiency of the antisense amplicon folding (stages A→B→C in Fig. 3). The cleavage is anticipated to be nearly quantitative even in the case of the unmodified amplicon (see below). However, the flap stabilization prompts the DNA polymerase to cut the duplex strand deeper toward the 3′-end of the hybridized flap. Consequently, this would lead to shorter 3′-flap fragments in the sense amplicons (stages D and E, Fig. 3). The 3′-flap truncation translates into the less stable self-extension complexes in stage H and then PCR termination in stage I (Fig. 3).
Asymmetric Snake PCR
The asymmetric PCR format22 was found to benefit the Snake assay.13 Indeed, as illustrated in Figure 5, the disproportional increase in the sense amplicon concentration stimulated by a fourfold excess of the reverse primer Pr2 noticeably improved the reaction signals. Unfortunately, the assay improvement did not apply to the detection sensitivity. For many of the 14-mer flap primers studied, the threshold values actually increased, reflecting a decline in the PCR efficiency (compare asymmetric vs. symmetric two-step PCR, Table 1). In theory, disequilibrium in the primer concentrations should have no effect on the DNA replication yield, unless the primer used in limited amounts becomes kinetically disadvantaged and cannot complete its hybridization and extension within a given cycle period. The observed negative effect on PCR efficiency is likely to be an attribute of the relatively long 14-mer flap sequences. For example, use of the very short 6-8-mer flaps (see below, Table 2) had the opposite effect on PCR and the improvements are most likely due to overstimulation of the signal-producing mechanism through the A→B→C→D pathway shown in Figure 4.
Fig. 5.
Fig. 5.
Real-time PCR detection of the β2-microglobulin target sequence in TaqMan and Snake assays. Shown are the reactions using one of the best-performing Snake primers (Pr3c) carrying a 14-mer 5′-flap sequence. Experiments with other analogs (more ...)
Table 2.
Table 2.
Effect of Nucleotide Modifications in Polymerase Chain Reaction Amplicons and Short 6-8-Mer 5′-Flap Sequences of Snake Primers on Threshold Values (Ct) of Fluorescence Curves During Real-Time Snake Polymerase Chain Reaction
Three-Step PCR in the Snake Assay
PCR amplicons produced for diagnostic purposes are commonly short and rarely exceed 150–200 base pairs in length. Amplification yields of these DNA fragments are usually good, which allows the use of simplified two-step PCR, wherein the primer annealing and extension are combined into one step. The three-step PCR—with the annealing and extension steps divided and performed at different temperatures—usually provides no advantage to nucleic acids detection. For example, incorporation of an additional extension step (15 s at 72°C) into the otherwise standard symmetric two-step PCR had no effect on the TaqMan assay (Ct2-step = 23.1 ± 0.2 vs. Ct3-step = 23.0 ± 0.3). Once again, Snake was found to be different in this aspect. Considerable improvement in PCR efficiency was observed for all derivatives of the 14-mer flap primer Pr3a-i (compare the data for two-step vs. three-step asymmetric PCR, Table 1). Despite the long 14-mer flaps and all the problems associated with them, in a few cases the Snake system matched or even outperformed the TaqMan competitor in the detection sensitivity (e.g., primer Pr3h with ΔCt =  0.9).
The reason for such a profoundly positive Snake system response to the three-step PCR protocol is actually well understood. Raising the reaction temperature to 72°C melts all of the amplicon secondary structures and, in particular, the sense amplicon folding. The generic primers Pr1 and Pr2 were found to perform very well at 72°C. Addition of the 5′-flap sequences in Snake further improved the primer hybridization properties. At the elevated temperatures of 72°C–75°C, Taq polymerase is known to exhibit maximum activity, incorporating more than 60 nucleotides per second.23,24 All of these factors lead to the completion of the DNA replication in a short period (e.g., 15 s). The base pair destabilization of 5′-flap sequences in Snake primers solves the problem of the antisense amplicon folding (Fig. 3), whereas the three-step PCR protocol effectively addresses the sense amplicon issues (Fig. 4). Not surprisingly, the combination of these approaches was found to be the most effective (primers Pr3b-d and Pr3h in two-step symmetric vs. three-step asymmetric PCR, Table 1).
Use of Very Short 6-8-Mer 5′-Flap Primer Sequences in Real-Time Snake PCR
The 6-8-mer flap sequences in forward primers Pr4-Pr6 (Fig. 2) are too short to form stable secondary structures in Snake amplicons at the standard reaction conditions.13 Two measures were taken to address the issue. First, the magnesium chloride concentration was increased from 2 to 5 mM. Second, the annealing temperature was reduced from 64°C to 56°C. These protocol modifications also allowed the use of a shorter 12-mer FRET probe Pb3 with no loss in the signal productivity. The results of this study are summarized in Table 2.
Similar to the primers with long 14-mer flap sequences, the Snake assays employing primers with short 6-8-mer flaps positively responded to the duplex-destabilization (compare Ct values obtained for primers Pr4a-6a vs. Pr4b-6b using symmetric two-step PCR, Table 2). An average delay in the threshold values obtained for primers Pr4-Pr6 (symmetric two-step PCR, Table 2) was greater than that observed for the 14-mer flap primers Pr3a-i (symmetric two-step PCR, Table 1). However, as can be seen in Figure 6, this result was due to the exceptionally poor signal generated in the cases of short 6-8-mer flaps. In contrast to the 14-mer flap primers (Fig. 5), asymmetry in the primer concentrations was the major factor in restoring the fluorescence signal back to the normal level (Fig. 6), whereas introduction of the additional extension stage in three-step PCR had a positive, but very minor effect (Fig. 6 and Table 2). The difference in the system performance between symmetric and asymmetric PCR was very significant, and this allowed the Snake assays with short 6-8-mer flap primers to match or even outperform the conventional TaqMan assay in detection sensitivity.
Fig. 6.
Fig. 6.
Effect of changes in PCR protocol on real-time performance of Snake assays employing very short 6-mer 5′-flap sequence in the forward primer Pr6a. The primers and FRET probes used are indicated for each assay, and their structures are shown Figure (more ...)
Base Modification of PCR Products for Specific Stabilization of the Catalytic Sense Amplicon Folding
There are two mutually exclusive approaches to improve the hybridization properties of PCR oligonucleotides. The first is to modify the primers and probes. The toolbox includes peptide nucleic acid (PNA),25 locked nucleic acid (LNA),26,27 conjugated minor groove binders,28,29 intercalators,30,31 and certain base modifications.32 The second approach is based on modification of the detected target DNAs, and it can be as potent in duplex stabilization as the traditional approaches cited above. Amplicons with enhanced hybridization properties can be produced in PCR using base-modified duplex-stabilizing analogs of natural deoxynucleoside 5′-triphosphates.33 This method was found to be very effective in Snake—specifically for the assays employing the primers with very short 6-8-mer flap sequences—and the results are summarized in Table 2.
The biggest improvement in the threshold values was observed in the cases where d(2-amA)TP was completely substituted for dATP. The effect of the d(5-PrU)TP analog was smaller, and there is a reason for that. The 6-8-mer flap sequences happened to be A-rich. These flaps do not incorporate thymidines. Because of that, application of d(2-amA)TP had no effect on the antisense amplicon folding, but efficiently stabilized the sense secondary structure. For the same reason, the d(5-PrU)TP analog stabilized both amplicon foldings, sense and antisense equally well. Particularly, the stabilization of the antisense secondary structure in the case of d(5-PrU)TP is suspected to cause the minor increase in threshold values in comparison to the same reactions but stimulated with the d(2-amA)TP substitution (compare Ct and ΔCt values for primers Pr4a–Pr6a in natural vs. d(2-amA)TP vs. d(5-PrU)TP three-step asymmetric PCR, Table 2). This observation leads to an important rule regarding the optimal use of the d(2-amA)TP and d(5-PrU)TP analogs in Snake. When the flap sequences are A-rich, then the best analog to use is d(2-amA)TP, whereas for the T-rich sequences the most effective analog is d(5-PrU)TP. In cases of mixed A/T-sequences, either dNTP analog can be applied, and the sense amplicon folding is still going to be preferentially stabilized compared to the antisense stem-loop structure.
This dNTP modification method can actually benefit any real-time assay33 because of the general improvement in hybridization properties of all primers and probes. In the present study, the approach allowed the use of exceptionally short 9-11-mer FRET probes (Pb4 and Pb5, Fig. 2). As can be seen in Figure 7, these probes produce very little or no signal in the conventional TaqMan assay. Apparently, the probes' hybridization properties are too weak to fulfill requirements of the TaqMan system (TmPb4 = 49°C and TmPb5 = 46°C, Fig. 2). The probes' insufficiency in hybridization, however, was not a problem in Snake, wherein the maximum signal and excellent detection sensitivity were demonstrated (CtPb4 = 20.6 ± 0.1 and CtPb5 = 21.4 ± 0.1).
Fig. 7.
Fig. 7.
Performance of exceptionally short 9 and 11-mer FRET probes in real-time Snake and TaqMan PCR. The TaqMan detection experiments were conducted using two-step symmetric PCR, regular forward and reverse primers (Pr1 and Pr2) and 9 or 11-mer FRET probe as (more ...)
Amplicon Repair in Snake
The FRET probe cleavage in TaqMan is not quantitative. For example, in the case of the TaqMan assay shown in Figure 5, the efficiency of cleavage was in the range of ~60%–70%.13 Because of the intermolecular complex formation, there is always a chance in TaqMan that primers will hybridize to amplicons and extend before the probe binds to its target. In contrast, intra-molecular duplex formation of the antisense amplicon (Fig. 3A) is very fast. In the case of the long 14-mer flap sequence (Pr3a, Fig. 2), the stem-loop secondary structure of the amplicon is very stable (Tm = 76°C) at the PCR annealing temperature of 64°C. Therefore, it is absolutely reasonable to expect the antisense amplicon cleavage to be very efficient (≥95%) and nearly complete. The cleaved antisense amplicon replicates and produces a truncated sense amplicon, which is useless in the signal generation (stage G, Fig. 3). Moreover, the same truncated sense amplicon can self-extend through the E→H→I pathway (Fig. 3) and terminate PCR. Nevertheless, despite all of these potentially catastrophic factors, the longest 14-mer flap primer Pr3a produced an excellent signal in the Snake assay. In addition, the antisense amplicon cleavage was found to be responsible only for half of the delay in threshold appearance (~2 cycles).13 This, in turn, means that the almost quantitative antisense amplicon cleavage resulted only in a ~12% drop in the PCR yield.
The remarkable resistance of the Snake system to the antisense amplicon cleavage can be explained by the amplicon repair process shown in Figure 3 (E→F pathway). Unless this amplicon repair is very efficient, it would be hard to explain otherwise the results shown in Figure 8. In this experiment, the Snake primer Pr3b was gradually replaced with a regular forward primer Pr1 that is homologous to Pr3b but has no 5′-flap (Fig. 2). As can be seen (Fig. 8), loss of the flap sequence up to 50% had no effect on the system performance and the real-time curves overlap. The negative changes began to appear only when the fraction of the 5′-flap primer reached 25%. This tolerance of the assay to the loss of the flap sequences means, in particular, that the quality control requirement for the manufacturing of Snake primers will remain the same as for any other PCR primer and this should expedite the technology's market penetration.
Fig. 8.
Fig. 8.
This experiment illustrates the resistance of the Snake detection system to loss of the 5′-flap sequence in the forward primer. The real-time curves are shown in a differential format for better detection of minor differences. The reactions were (more ...)
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.3436 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 (Fig. 3). 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 (Fig. 4).
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 (Fig. 3). 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 Tables 1 and and22).
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, Fig. 2) 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.2532
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, Fig. 3) 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.
Abbreviations
Ctcurve threshold value
2-amA2-amino deoxyadenosine
d(2-amA)TP2-amino deoxyadenosine 5′-triphosphate
dIdeoxyinosine
5-PrU5-propynyl-deoxyuridine
d(5-PrU)TP5-propynyl deoxyuridine 5′-triphosphate
dUdeoxyuridine
FRETFörster resonance energy transfer
LANLocked nucleic acid
SNPsingle nucleotide polymorphism
MGBminor groove binder
PCRpolymerase chain reaction
PNApeptide nucleic acid
TaqThermus aquaticus
Tmmelting temperature

Acknowledgments
This research project was funded by the National Institutes of Health (SBIR grant 1R43GM093446-01). Access to the necessary facilities and instrumentation and all reagents for the research were provided by Cepheid. The author thanks Cepheid's employees and managers, particularly John Bishop, David Persing, Humberto Reyes, Peter Dailey, John Smith, Vincent Powers, Alexander Gall, Sergey Lokhov, and Ekaterina Viazovkina, for interest, technical help, and support. The assistance of Ekaterina Kutyavin (University of Washington, Seattle, WA) and Vassily Kutyavin (Haverford College, Haverford, PA) in the editing and revision of the article is gratefully acknowledged.
Disclosure Statement
No competing financial interests exist.
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