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 language | English |
|---|---|
| Pages (from-to) | 105-111 |
| Number of pages | 7 |
| Journal | Analytical Biochemistry |
| Volume | 369 |
| Issue number | 1 |
| DOIs | |
| Publication status | Published - 2007 Oct 1 |
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Keywords
- Competitive PCR
- Fluorescence quenching
- Genetically modified organisms
- Quenching probe
- Real-time PCR
- RRS
ASJC Scopus subject areas
- Biochemistry
- Biophysics
- Molecular Biology
Cite this
Calibration-curve-free quantitative PCR : A quantitative method for specific nucleic acid sequences without using calibration curves. / Tani, Hidenori; Kanagawa, Takahiro; Morita, Nao; Kurata, Shinya; Nakamura, Kazunori; Tsuneda, Satoshi; Noda, Naohiro.
In: Analytical Biochemistry, Vol. 369, No. 1, 01.10.2007, p. 105-111.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Calibration-curve-free quantitative PCR
T2 - A quantitative method for specific nucleic acid sequences without using calibration curves
AU - Tani, Hidenori
AU - Kanagawa, Takahiro
AU - Morita, Nao
AU - Kurata, Shinya
AU - Nakamura, Kazunori
AU - Tsuneda, Satoshi
AU - Noda, Naohiro
PY - 2007/10/1
Y1 - 2007/10/1
N2 - 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.
AB - 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.
KW - Competitive PCR
KW - Fluorescence quenching
KW - Genetically modified organisms
KW - Quenching probe
KW - Real-time PCR
KW - RRS
UR - http://www.scopus.com/inward/record.url?scp=34548189670&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=34548189670&partnerID=8YFLogxK
U2 - 10.1016/j.ab.2007.06.047
DO - 10.1016/j.ab.2007.06.047
M3 - Article
C2 - 17679100
AN - SCOPUS:34548189670
VL - 369
SP - 105
EP - 111
JO - Analytical Biochemistry
JF - Analytical Biochemistry
SN - 0003-2697
IS - 1
ER -