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Single-channel multiplexing without melting curve analysis in real-time PCR.

Lee YJ, Kim D, Lee K, Chun JY - Sci Rep (2014)

Bottom Line: In this technique, we show the intensity of the fluorescence signals of two discrete Tm targets is different at certain temperatures called detection temperatures, by which a high Tm target can be detected regardless of a low Tm target.We then identify the low Tm target by utilizing a change of the fluorescence signals between two different detection temperatures.Furthermore, it enables us to determine quantification of each target in a single channel, possibly facilitating convenient patient care for drug treatment in clinics.

View Article: PubMed Central - PubMed

Affiliation: Seegene, Inc., Taewon Bldg., 91 Ogeum-ro, Songpa-Gu, Seoul 138-828, South Korea.

ABSTRACT
Multiplex real-time PCR with quantification of targets in a single fluorescence channel has been the demand in biotechnology industry. Here, we develop a novel analytical real-time PCR technique to detect multiple targets in a single fluorescence channel without melting curve analysis. In this technique, we show the intensity of the fluorescence signals of two discrete Tm targets is different at certain temperatures called detection temperatures, by which a high Tm target can be detected regardless of a low Tm target. We then identify the low Tm target by utilizing a change of the fluorescence signals between two different detection temperatures. Furthermore, it enables us to determine quantification of each target in a single channel, possibly facilitating convenient patient care for drug treatment in clinics.

Show MeSH
Intensity of the fluorescence signals depends on detection temperature.Detection temperature means a certain temperature where the unquenched fluorescence signal is measured at each cycle during real-time PCR reaction. If there are two targets representing different Tm profiles, the fluorescence signals between two targets would be different at certain detection temperatures (marked in gray). Therefore, these temperatures differentiate the high Tm target from the low Tm target, by which the high Tm target can be detected.
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f1: Intensity of the fluorescence signals depends on detection temperature.Detection temperature means a certain temperature where the unquenched fluorescence signal is measured at each cycle during real-time PCR reaction. If there are two targets representing different Tm profiles, the fluorescence signals between two targets would be different at certain detection temperatures (marked in gray). Therefore, these temperatures differentiate the high Tm target from the low Tm target, by which the high Tm target can be detected.

Mentions: During real-time PCR reaction, the duplex-driven fluorescence signal (designated as a yellow circle) of the probes depends on the temperature because of their melting curves (Fig. 1). Therefore, different intensities of those signals at certain temperatures at each cycle are able to detect the targets with different Tm values (highlighted in gray). These temperatures are defined as “detection temperatures” at which the intensity of a target's fluorescence signal is measured.


Single-channel multiplexing without melting curve analysis in real-time PCR.

Lee YJ, Kim D, Lee K, Chun JY - Sci Rep (2014)

Intensity of the fluorescence signals depends on detection temperature.Detection temperature means a certain temperature where the unquenched fluorescence signal is measured at each cycle during real-time PCR reaction. If there are two targets representing different Tm profiles, the fluorescence signals between two targets would be different at certain detection temperatures (marked in gray). Therefore, these temperatures differentiate the high Tm target from the low Tm target, by which the high Tm target can be detected.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4262828&req=5

f1: Intensity of the fluorescence signals depends on detection temperature.Detection temperature means a certain temperature where the unquenched fluorescence signal is measured at each cycle during real-time PCR reaction. If there are two targets representing different Tm profiles, the fluorescence signals between two targets would be different at certain detection temperatures (marked in gray). Therefore, these temperatures differentiate the high Tm target from the low Tm target, by which the high Tm target can be detected.
Mentions: During real-time PCR reaction, the duplex-driven fluorescence signal (designated as a yellow circle) of the probes depends on the temperature because of their melting curves (Fig. 1). Therefore, different intensities of those signals at certain temperatures at each cycle are able to detect the targets with different Tm values (highlighted in gray). These temperatures are defined as “detection temperatures” at which the intensity of a target's fluorescence signal is measured.

Bottom Line: In this technique, we show the intensity of the fluorescence signals of two discrete Tm targets is different at certain temperatures called detection temperatures, by which a high Tm target can be detected regardless of a low Tm target.We then identify the low Tm target by utilizing a change of the fluorescence signals between two different detection temperatures.Furthermore, it enables us to determine quantification of each target in a single channel, possibly facilitating convenient patient care for drug treatment in clinics.

View Article: PubMed Central - PubMed

Affiliation: Seegene, Inc., Taewon Bldg., 91 Ogeum-ro, Songpa-Gu, Seoul 138-828, South Korea.

ABSTRACT
Multiplex real-time PCR with quantification of targets in a single fluorescence channel has been the demand in biotechnology industry. Here, we develop a novel analytical real-time PCR technique to detect multiple targets in a single fluorescence channel without melting curve analysis. In this technique, we show the intensity of the fluorescence signals of two discrete Tm targets is different at certain temperatures called detection temperatures, by which a high Tm target can be detected regardless of a low Tm target. We then identify the low Tm target by utilizing a change of the fluorescence signals between two different detection temperatures. Furthermore, it enables us to determine quantification of each target in a single channel, possibly facilitating convenient patient care for drug treatment in clinics.

Show MeSH