Calibration-curve-free quantitative PCR: A quantitative method for specific nucleic acid sequences without using calibration curves

Hidenori Tani, Takahiro Kanagawa, Nao Morita, Shinya Kurata, Kazunori Nakamura, Satoshi Tsuneda, Naohiro Noda

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

We have developed a simple quantitative method for specific nucleic acid sequences without using calibration curves. This method is based on the combined use of competitive polymerase chain reaction (PCR) and fluorescence quenching. We amplified a gene of interest (target) from DNA samples and an internal standard (competitor) with a sequence-specific fluorescent probe using PCR and measured the fluorescence intensities before and after PCR. The fluorescence of the probe is quenched on hybridization with the target by guanine bases, whereas the fluorescence is not quenched on hybridization with the competitor. Therefore, quench rate (i.e., fluorescence intensity after PCR divided by fluorescence intensity before PCR) is always proportional to the ratio of the target to the competitor. Consequently, we can calculate the ratio from quench rate without using a calibration curve and then calculate the initial copy number of the target from the ratio and the initial copy number of the competitor. We successfully quantified the copy number of a recombinant DNA of genetically modified (GM) soybean and estimated the GM soybean contents. This method will be particularly useful for rapid field tests of the specific gene contamination in samples.

Original languageEnglish
Pages (from-to)105-111
Number of pages7
JournalAnalytical Biochemistry
Volume369
Issue number1
DOIs
Publication statusPublished - 2007 Oct 1

Keywords

  • Competitive PCR
  • Fluorescence quenching
  • Genetically modified organisms
  • Quenching probe
  • RRS
  • Real-time PCR

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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