Real-time PCR reactions involve two processes that simultaneously occur and are mechanistically inter-dependent: target amplification and signal generation. It is the combination of these two processes that enables real-time monitoring of PCR product accumulation in a closed-tube format providing the basis for sensitive and reproducible quantification of input samples. Real-time PCR assay design starts with selection of target region and primer sequences as well as optimization of amplification efficiency including reagent composition, concentration and cycling conditions. Signal generation during PCR is generally achieved through hybridization of a fluorescent probe resulting in differential fluorescent signal incurred by the binding or cleavage of the probe. Multiple factors have to be considered when designing a probe within the amplicon region: (i) optimizing sensitivity for detection of low level targets, (ii) maximizing mismatch tolerance or discrimination while not adversely affecting detection of perfect-match sequences and (iii) lowering pre-amplification background signals and improving signal to noise ratio. Optimization of the probe is usually focused on either selecting an optimal probe-binding site or modulating hybridization stability once the former is determined. Binding site choices are often limited by the primer selection and the distribution of mismatches in the target region. Thus, thermodynamic modulation of the probe may further improve the performance attributes of a real-time PCR assay.
Several previous studies have investigated the relationship between the thermodynamic properties of different probes and their ability to differentiate between mismatch and perfect-match sequences (27
). However, the benefits derived from these probe designs are potentially undermined by the inefficient binding to perfect-match sequence because of the intrinsically interdependent and competitive behaviors of the probes. Recently introduced DNA conjugate or nucleoside analogues (e.g. MGB, LNA and PNA) can significantly enhance binding affinity of short probes against perfect match sequences and confer increased mismatch discrimination (39–43
). Similar to the above-mentioned approaches, focus was often placed on one particular outcome of real-time PCR (i.e. mismatch tolerance or discrimination), yet assays are almost always designed to meet multiple specifications. Here, we report the development of a partially double-stranded linear DNA probe. Many features of this novel probe design can be thermodynamically modulated with considerable independence. Therefore, the probe can be designed such that an individual performance feature can be improved while others are not negatively impacted.
Fluorescent signal of HP, in the absence of QO or target DNA, displayed a single-phase transition when temperature decreased, presumably due to spontaneous coiling of the oligonucleotide that brings quencher and fluorophore into close proximity (13
). The probability of quenching at each temperature and the signal level during transition is most likely dependent on the distance between the two ends of the primary sequence. Indeed, while HP20 had a θm
of ~32°C and was maximally quenched at ~20°C, HP31 was quenched by only ~50% at 20°C (). Since random coiling is also affected by the rigidity of a single-stranded hybridization probe, the actual correlation between melting stability and oligonucleotide length observed in this study may be specific to the chosen oligo length range, sequences, labeling position and divalent cation concentration (13
). The quenched signal of HP measured in a melting analysis should be equal to the pre-amplification background signal in a real-time PCR with all other conditions being identical. Data presented in this study demonstrated that the background fluorescence can be effectively adjusted with QO through thermodynamic modulation of double-stranded DNA probe stability, as shown in .
Thermodynamic modulation of the probe performance is essentially adjusting the balance between two intrinsically competitive processes: binding of the fluorescent-labeled HP either to the target or to the shorter QO. Since the shorter strand of the DNA duplex determines its hybridization stability, target binding is always more stable with longer HP within a practical range. Similarly, longer QO and higher concentration increases double-stranded probe stability and competes with target for probe binding. Because the design strategy introduced in this study stipulates a longer HP than QO, the HP preferentially binds to the target unless destabilized by the presence of mismatches. When the destabilization is sufficiently large, the level of mismatch tolerance or discrimination can be modulated by the length and/or concentration of the QO.
The effectiveness of this modulation depends on the relative thermodynamic stability between the two processes, as measured by θm. When θm of probe melting (θm,HP:QO) is significantly lower than that of target binding (θm,HP:Target), changing probe duplex stability has little impact on target binding and signal generation, as shown in . To the contrary, when θm,HP:QO is significantly higher than θm,HP:Target especially when mismatches are present, the fluorescent signal specific for HP:Target becomes smaller [e.g. HP20:QO16 (1:3) with 1 mismatch target shown in ]. Therefore, desirable mismatch performance can be achieved by adjusting θm for probe binding relative to target binding. Specifically, setting the probe θm above that of mismatch target binding and below that of perfect-match target binding results in mismatch discrimination. Setting the probe θm below that of both mismatch and perfect-match target binding results in mismatch tolerance. This was shown experimentally when HP20 without a quencher oligo was compared with HP20 with quencher oligo of 16 bases. HP20 alone with a lower θm (30°C) than both perfect-match (61°C) and mismatch targets (52°C) tolerated a single mismatch whereas HP20:QO16 (1:3; θm = 55°C) discriminated between perfect-match and mismatch targets (compare A, B with E, F). Similarly, while a single mismatch did not significantly affect binding of HP31 in the presence of QO16 (I, J), three mismatches in the probe-binding region were sufficient to destabilize the binding (; melting data not shown). Under the condition where θm,HP:QO is significantly higher than θm,HP:Target, modulating θm for each binding process by adjusting HP length, QO length, or changing relative concentration between QO and HP will also change the signal gain. The extent of mismatch discrimination can thus be adjusted with precision, which was demonstrated by the comparison between HP20:QO16 and HP31:QO16 (C and I), between HP20:QO16 and HP20:QO12 (E and G) and between 1:1 and 1:3 conditions for HP20:QO16 (C and E).
In addition to the consideration of length and concentration of HP and QO, PCR read temperature is another factor that can have a fundamental impact on assay performance. The thermodynamic behaviors of a probe such as target-binding and mismatch tolerance or discrimination are driven by specific detection temperatures. For a given probe design, PCR reactions can be detected at a temperature where there is differential binding between perfect-match and mismatch targets to achieve mismatch discrimination (A, B). Alternatively, a lower read temperature where both mismatch and perfect-match targets bind efficiently to the probe can be used to achieve mismatch tolerance and accurate quantification of diverse sequences (C, D).
The relative independence between HP-QO and HP-target binding and between binding to perfect-match and mismatch sequences makes the thermodynamic modulation particularly straightforward for partially double-stranded linear DNA probes. In this probe system, HP can be lengthened or shortened for mismatch tolerance or discrimination without impacting HP:QO stability, as shown in . Moreover, QO length and HP:QO ratio can be adjusted specifically for mismatch tolerance or discrimination without impacting HP binding to perfect-match targets, as shown in and and . This important characteristic adds considerable flexibility and distinguishes partially double-stranded linear DNA probes from other previously described probes. Molecular beacons bind to targets with high specificity due to the formation of stem-loop structure that competes with target binding. Molecular beacons are thus very sensitive to nucleotide variations and can be conveniently optimized for differentiating sequence polymorphisms. However, modulating molecular beacons to tolerate sequence variations is not straightforward because lengthening the loop or weakening the stem can potentially increase background signal and affect sensitivity. TaqMan technology utilizes conditions that are also highly stringent for mismatches in the target-binding region due to the relatively high polymerase extension temperature required for cleavage and signal generation, making it potentially unsuitable for detection of polymorphic sequences. Symmetric or near-symmetric double-stranded probes have been designed for maximal mismatch discrimination (34
). However, they were also shown to suffer in binding kinetics for perfect-match targets (34
). Compared with these probe technologies, the partially double-stranded linear DNA probe introduced in this study has the following advantages: (i) ease of design by independently modulating HP and QO, (ii) design flexibility for either mismatch tolerance or discrimination, (iii) ability to modulate probe performance by titration of the quencher oligo, without changing oligo length or nucleotide composition and (iv) detection temperature uncoupled from extension step allowing additional control over mismatch stringency.
In conclusion, we introduced in this study a unique and versatile partially double-stranded linear DNA probe design strategy for real-time PCR application. We demonstrated the feasibility of thermodynamic modulation of two probe strands to meet the complex performance requirements for diagnostic assay development. The concept of using a melting model for probe design in a PCR reaction is widely applicable when probe hybridization is involved in a solution-based reaction. The combination of two independent oligonucleotide strands allows for easier optimization of assay performance. Partially double-stranded DNA probes can be modulated to either discriminate mismatches for genotyping or SNP detection or tolerate mismatches as demonstrated with detection of diverse HIV-1 sequences. The mismatch tolerance of this novel class of probes has been demonstrated by several evaluations of Abbott RealTim
e HIV-1 assay on genetically diverse specimens (9