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Multicolor combinatorial probe coding for real-time PCR.

Huang Q, Zheng L, Zhu Y, Zhang J, Wen H, Huang J, Niu J, Zhao X, Li Q - PLoS ONE (2011)

Bottom Line: MCPC was then improved from a one-primer-pair setting to a multiple-primer-pair format through Homo-Tag Assisted Non-Dimer (HAND) system to allow multiple primer pairs to be included in one reaction.This improvement was demonstrated via identification of one of the possible 10 foodborne pathogen candidates with 10 pairs of primers included in one reaction, which had limit of detection equivalent to the uniplex PCR.MCPC strategy could expand the scope of real-time PCR assays in applications which are unachievable by current labeling strategy.

View Article: PubMed Central - PubMed

Affiliation: Engineering Research Centre of Molecular Diagnostics of the Ministry of Education, Department of Biomedical Sciences and the Key Laboratory of Cell Biology and Tumor Cell Engineering of the Ministry of Education, School of Life Sciences, Xiamen University, Xiamen, China.

ABSTRACT
The target volume of multiplex real-time PCR assays is limited by the number of fluorescent dyes available and the number of fluorescence acquisition channels present in the PCR instrument. We hereby explored a probe labeling strategy that significantly increased the target volume of real-time PCR detection in one reaction. The labeling paradigm, termed "Multicolor Combinatorial Probe Coding" (MCPC), uses a limited number (n) of differently colored fluorophores in various combinations to label each probe, enabling one of 2(n)-1 genetic targets to be detected in one reaction. The proof-of-principle of MCPC was validated by identification of one of each possible 15 human papillomavirus types, which is the maximum target number theoretically detectable by MCPC with a 4-color channel instrument, in one reaction. MCPC was then improved from a one-primer-pair setting to a multiple-primer-pair format through Homo-Tag Assisted Non-Dimer (HAND) system to allow multiple primer pairs to be included in one reaction. This improvement was demonstrated via identification of one of the possible 10 foodborne pathogen candidates with 10 pairs of primers included in one reaction, which had limit of detection equivalent to the uniplex PCR. MCPC was further explored in detecting combined genotypes of five β-globin gene mutations where multiple targets were co-amplified. MCPC strategy could expand the scope of real-time PCR assays in applications which are unachievable by current labeling strategy.

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Schematic representation of the Multicolor Combinatorial Probe Coding (MCPC) strategy for multiplex, real-time PCR genotyping using displacing probes.Four differently colored fluorophores (FAM: green; HEX: orange; ROX: red; and Cy5: blue) and one universal quencher (DABCYL: black) are used to depict the principal of MCPC. A) Illustration of how half the molecules of a third probe in an assay can be labeled in one color and the other half of the molecules of the third probe can be labeled with a second color, producing a single probe that generates a two-color signal when it hybridizes to its target. B) Four probes, each labeled with a different single color. C) Six probes, each labeled with a unique combination of two of the four colors. D) Four probes, each labeled with a unique combination of three of the four colors, and one probe labeled with all four colors. For each fluorophore, a unique four-digit code, MCPC signature, is given, i.e., FAM is 1000, HEX is 0100, ROX is 0010, Cy5 is 0001. For the combined fluorophores, the MCPC signatures are also combined for coding.
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pone-0016033-g001: Schematic representation of the Multicolor Combinatorial Probe Coding (MCPC) strategy for multiplex, real-time PCR genotyping using displacing probes.Four differently colored fluorophores (FAM: green; HEX: orange; ROX: red; and Cy5: blue) and one universal quencher (DABCYL: black) are used to depict the principal of MCPC. A) Illustration of how half the molecules of a third probe in an assay can be labeled in one color and the other half of the molecules of the third probe can be labeled with a second color, producing a single probe that generates a two-color signal when it hybridizes to its target. B) Four probes, each labeled with a different single color. C) Six probes, each labeled with a unique combination of two of the four colors. D) Four probes, each labeled with a unique combination of three of the four colors, and one probe labeled with all four colors. For each fluorophore, a unique four-digit code, MCPC signature, is given, i.e., FAM is 1000, HEX is 0100, ROX is 0010, Cy5 is 0001. For the combined fluorophores, the MCPC signatures are also combined for coding.

Mentions: To enable a large number of probes to be labeled, and many targets to be distinguished with a limited number of fluorophores and fluorescence detection channels, we used differently colored fluorophores in various combinations to label individual probes. For example, two differently colored fluorophores can be used to distinguishably label three different probes, i.e., two probes can each be labeled with one color, a third probe can be labeled with both colors. If four different fluorophores can be used, four probes can each be labeled with a unique (single) color, six probes can each be labeled with a unique combination of two of the four colors, four probes can each be labeled with a unique combination of three of the four colors, and one probe can be labeled with all four colors, resulting in a total of 15 uniquely labeled probes (Figure 1).


Multicolor combinatorial probe coding for real-time PCR.

Huang Q, Zheng L, Zhu Y, Zhang J, Wen H, Huang J, Niu J, Zhao X, Li Q - PLoS ONE (2011)

Schematic representation of the Multicolor Combinatorial Probe Coding (MCPC) strategy for multiplex, real-time PCR genotyping using displacing probes.Four differently colored fluorophores (FAM: green; HEX: orange; ROX: red; and Cy5: blue) and one universal quencher (DABCYL: black) are used to depict the principal of MCPC. A) Illustration of how half the molecules of a third probe in an assay can be labeled in one color and the other half of the molecules of the third probe can be labeled with a second color, producing a single probe that generates a two-color signal when it hybridizes to its target. B) Four probes, each labeled with a different single color. C) Six probes, each labeled with a unique combination of two of the four colors. D) Four probes, each labeled with a unique combination of three of the four colors, and one probe labeled with all four colors. For each fluorophore, a unique four-digit code, MCPC signature, is given, i.e., FAM is 1000, HEX is 0100, ROX is 0010, Cy5 is 0001. For the combined fluorophores, the MCPC signatures are also combined for coding.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3021529&req=5

pone-0016033-g001: Schematic representation of the Multicolor Combinatorial Probe Coding (MCPC) strategy for multiplex, real-time PCR genotyping using displacing probes.Four differently colored fluorophores (FAM: green; HEX: orange; ROX: red; and Cy5: blue) and one universal quencher (DABCYL: black) are used to depict the principal of MCPC. A) Illustration of how half the molecules of a third probe in an assay can be labeled in one color and the other half of the molecules of the third probe can be labeled with a second color, producing a single probe that generates a two-color signal when it hybridizes to its target. B) Four probes, each labeled with a different single color. C) Six probes, each labeled with a unique combination of two of the four colors. D) Four probes, each labeled with a unique combination of three of the four colors, and one probe labeled with all four colors. For each fluorophore, a unique four-digit code, MCPC signature, is given, i.e., FAM is 1000, HEX is 0100, ROX is 0010, Cy5 is 0001. For the combined fluorophores, the MCPC signatures are also combined for coding.
Mentions: To enable a large number of probes to be labeled, and many targets to be distinguished with a limited number of fluorophores and fluorescence detection channels, we used differently colored fluorophores in various combinations to label individual probes. For example, two differently colored fluorophores can be used to distinguishably label three different probes, i.e., two probes can each be labeled with one color, a third probe can be labeled with both colors. If four different fluorophores can be used, four probes can each be labeled with a unique (single) color, six probes can each be labeled with a unique combination of two of the four colors, four probes can each be labeled with a unique combination of three of the four colors, and one probe can be labeled with all four colors, resulting in a total of 15 uniquely labeled probes (Figure 1).

Bottom Line: MCPC was then improved from a one-primer-pair setting to a multiple-primer-pair format through Homo-Tag Assisted Non-Dimer (HAND) system to allow multiple primer pairs to be included in one reaction.This improvement was demonstrated via identification of one of the possible 10 foodborne pathogen candidates with 10 pairs of primers included in one reaction, which had limit of detection equivalent to the uniplex PCR.MCPC strategy could expand the scope of real-time PCR assays in applications which are unachievable by current labeling strategy.

View Article: PubMed Central - PubMed

Affiliation: Engineering Research Centre of Molecular Diagnostics of the Ministry of Education, Department of Biomedical Sciences and the Key Laboratory of Cell Biology and Tumor Cell Engineering of the Ministry of Education, School of Life Sciences, Xiamen University, Xiamen, China.

ABSTRACT
The target volume of multiplex real-time PCR assays is limited by the number of fluorescent dyes available and the number of fluorescence acquisition channels present in the PCR instrument. We hereby explored a probe labeling strategy that significantly increased the target volume of real-time PCR detection in one reaction. The labeling paradigm, termed "Multicolor Combinatorial Probe Coding" (MCPC), uses a limited number (n) of differently colored fluorophores in various combinations to label each probe, enabling one of 2(n)-1 genetic targets to be detected in one reaction. The proof-of-principle of MCPC was validated by identification of one of each possible 15 human papillomavirus types, which is the maximum target number theoretically detectable by MCPC with a 4-color channel instrument, in one reaction. MCPC was then improved from a one-primer-pair setting to a multiple-primer-pair format through Homo-Tag Assisted Non-Dimer (HAND) system to allow multiple primer pairs to be included in one reaction. This improvement was demonstrated via identification of one of the possible 10 foodborne pathogen candidates with 10 pairs of primers included in one reaction, which had limit of detection equivalent to the uniplex PCR. MCPC was further explored in detecting combined genotypes of five β-globin gene mutations where multiple targets were co-amplified. MCPC strategy could expand the scope of real-time PCR assays in applications which are unachievable by current labeling strategy.

Show MeSH
Related in: MedlinePlus