Universal Quenching Probe System: Flexible, Specific, and Cost-Effective Real-Time Polymerase Chain Reaction Method

• Published in: Analytical chemistry (Washington) Vol. 81; no. 14; pp. 5678 - 5685
• Main Authors: Tani, Hidenori, Miyata, Ryo, Ichikawa, Kouhei, Morishita, Soji, Kurata, Shinya, Nakamura, Kazunori, Tsuneda, Satoshi, Sekiguchi, Yuji, Noda, Naohiro
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 15.07.2009
• Subjects: Analytical chemistry; Base Sequence; Chemistry; Electron transfer reactions; Exact sciences and technology; Fluorescence; Fluorescent Dyes - metabolism; Genotype; Genotype & phenotype; Humans; Nucleic Acid Denaturation; Nucleic Acid Probes - genetics; Nucleic Acid Probes - metabolism; Oligonucleotides - genetics; Oligonucleotides - metabolism; Polymerase Chain Reaction - economics; Polymerase Chain Reaction - methods; Polymorphism; Polymorphism, Single Nucleotide; Reaction kinetics; Spectrometric and optical methods; Time Factors; Transition Temperature; Dyes; Electron transfer; Real time; Guanine; Fluorescence spectrometry; Polymerase chain reaction; Efficiency; Fluorescent labelling; Luminescence quenching; Oligonucleotide; Single nucleotide polymorphism; Nucleic acid; Quantitative analysis
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac900414u

We have developed a flexible, specific, and cost-effective real-time polymerase chain reaction (PCR) method. In this technique, a quenching probe (QProbe) and a nonfluorescent 3′-tailed probe are used. The QProbe is a singly labeled oligonucleotide bearing a fluorescent dye that is quenched via electron transfer between the dye and a guanine base at a particular position. The nonfluorescent 3′-tailed probe consists of two parts: one is the target-specific sequence on the 5′ side, and the other is complementary to the QProbe on the 3′ side. When the QProbe/nonfluorescent 3′-tailed probe complex hybridizes with the target in PCR, the fluorescence of the dye is quenched. Fluorescence quenching efficiency is proportional to the amount of the target. We called this method the universal QProbe system. This method substantially reduces the cost of real-time PCR setup because the same QProbe can be used for different target sequences. Moreover, this method allows accurate quantification even in the presence of nonspecific PCR products because the use of nonfluorescent 3′-tailed probe significantly increases specificity. Our results demonstrate that this method can accurately and reproducibly quantify specific nucleic acid sequences in crude samples, comparable with conventional TaqMan chemistry. Furthermore, this method is also applicable to single-nucleotide polymorphism (SNP) genotyping.