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

Kennedy, Brett, Arar, Khalil, Reja, Valin and Henry, Robert J. (2006) Locked nucleic acids for optimizing displacement probes for quantitative real-time PCR. Analytical Biochemistry, 348 2: 294-299. doi:10.1016/j.ab.2005.10.037

Author Kennedy, Brett
Arar, Khalil
Reja, Valin
Henry, Robert J.
Title Locked nucleic acids for optimizing displacement probes for quantitative real-time PCR
Journal name Analytical Biochemistry   Check publisher's open access policy
ISSN 0003-2697
Publication date 2006-01
Sub-type Article (original research)
DOI 10.1016/j.ab.2005.10.037
Volume 348
Issue 2
Start page 294
End page 299
Total pages 6
Place of publication Maryland Heights, MO, United States
Publisher Academic Press
Language eng
Formatted 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.
Keyword Lna
Displacement Probes
Real-time Pcr
Duplex Probe
Complex Probe
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Non-UQ

Document type: Journal Article
Sub-type: Article (original research)
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Created: Mon, 07 Mar 2011, 16:11:14 EST