Universal quenching probe system: Flexible, specific, and cost-effective real-time polymerase chain reaction method

Hidenori Tani, Ryo Miyata, Kouhei Ichikawa, Soji Morishita, Shinya Kurata, Kazunori Nakamura, Satoshi Tsuneda, Yuji Sekiguchi, Naohiro Noda

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

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 non-fluorescent 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.

Original languageEnglish
Pages (from-to)5678-5685
Number of pages8
JournalAnalytical chemistry
Volume81
Issue number14
DOIs
Publication statusPublished - 2009 Jul 15

ASJC Scopus subject areas

  • Analytical Chemistry

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    Tani, H., Miyata, R., Ichikawa, K., Morishita, S., Kurata, S., Nakamura, K., Tsuneda, S., Sekiguchi, Y., & Noda, N. (2009). Universal quenching probe system: Flexible, specific, and cost-effective real-time polymerase chain reaction method. Analytical chemistry, 81(14), 5678-5685. https://doi.org/10.1021/ac900414u