Locked nucleic acids for optimizing displacement probes for quantitative real-time PCR
Section snippets
Design of primers and probes
Primers gapdh F1 and gapdh R1 and hydrolysis probe gapdh DLFP were designed using Beacon Designer 2 (Premier Biosoft) (Table 1). Conversion of the gapdh DLFP probe to displacement probes was performed according to the recommendations of Li and coworkers [8]. The probe strand was labeled with FAM at the 5′ end, whereas the quencher strand was labeled with BHQ-1 at the 3′ end and was designed to be four bases shorter than the probe strand. Probes were named with the probe strand first and the
Effect of LNA on probe–quencher complex Tm
To demonstrate the effect of LNA additions to the probe and quencher strands on the Tm of the probe complex, melting curve experiments were performed on all of the probes (Fig. 2). LNA has a significant effect on the probe complex Tm, with probe P1Q having a 9.7 °C higher Tm than probe PQ that differ only in the addition of three LNA bases. Probe P2Q1 is an 11 mer that has approximately the same Tm as probe PQ (21 mer) through the addition of six LNA bases in the probe and two LNA bases in the
Discussion
Real-time PCR detection using probe-based systems offers the advantage of improved specificity and sensitivity when compared with nonspecific detection using double-stranded DNA binding dyes such as SYBR Green I [7]. Further developments of novel probe technologies have the potential to facilitate the production of high-performance probes for high-throughput real-time PCR applications using standardized protocols without requiring extensive individual optimization.
Acknowledgments
The authors thank Agnelo Furtado and Dan Waters for assistance with manuscript preparation. We also thank the Australian Research Council for financial support.
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