Elsevier

Analytical Biochemistry

Volume 348, Issue 2, 15 January 2006, Pages 294-299
Analytical Biochemistry

Locked nucleic acids for optimizing displacement probes for quantitative real-time PCR

https://doi.org/10.1016/j.ab.2005.10.037Get rights and content

Abstract

Displacement probes have recently been described as a novel probe-based detection system for use in both quantitative real-time polymerase chain reaction (PCR) and single nucleotide polymorphism genotyping analysis. Previous reports have shown that shorter probes (23 mer) had improved detection sensitivity relative to longer probes (29 mer), with the likely reason for this effect being the improved hybridization kinetics of shorter probes. Sterically modified locked nucleic acids (LNAs) have been used to improve the design of a range of real-time PCR probes by raising the melting temperature (Tm) of the probe and enabling shorter probe designs to be considered. A displacement probe for gapdh was designed and tested successfully, and this probe was then redesigned with LNAs to an 11 mer probe. This probe showed increased detection sensitivity compared with the original 26 mer probe. To detect the widest range of displacement probe designs at maximum sensitivity, we have also developed a novel fluorescence capture two-step PCR protocol. This method produces enhanced probe quenching with a single standardized protocol ideal for high-throughput applications. The displacement probes tested produced sensitive and efficient quantitative analyses of template serial dilutions when compared with a range of commercially available predesigned real-time PCR detection systems, including TaqMan MGB probes, QuantiTect MGB probes, and LUX primers.

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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