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Related Concept Videos

Real Time RT-PCR02:57

Real Time RT-PCR

Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...
Labeling DNA Probes03:31

Labeling DNA Probes

DNA probes are fragments of DNA labeled with a reporter tag to enable their detection or purification. The resulting labeled DNA probes can then hybridize to target nucleic acid sequences through complementary base-pairing, and may be used to recover or identify these regions.
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...
PCR - Polymerase Chain Reaction01:32

PCR - Polymerase Chain Reaction

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PCR01:32

PCR

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Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella
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Multicolor combinatorial probe coding for real-time PCR.

Qiuying Huang1, Linlin Zheng, Yumei Zhu

  • 1Engineering 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.

Plos One
|January 26, 2011
PubMed
Summary
This summary is machine-generated.

Multiplex real-time PCR assays can now detect more targets using Multicolor Combinatorial Probe Coding (MCPC). This novel probe labeling strategy significantly expands detection capabilities in a single reaction.

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Genetics

Background:

  • Multiplex real-time PCR assays are limited by the number of available fluorescent dyes and instrument channels.
  • Existing labeling strategies restrict the number of detectable targets in a single reaction.

Purpose of the Study:

  • To develop a novel probe labeling strategy to significantly increase the number of detectable targets in multiplex real-time PCR.
  • To validate and improve this strategy for broader applications in genetic analysis.

Main Methods:

  • Developed Multicolor Combinatorial Probe Coding (MCPC) using combinations of fluorophores to label probes.
  • Validated MCPC by detecting 15 human papillomavirus types in one reaction.
  • Integrated MCPC with the Homo-Tag Assisted Non-Dimer (HAND) system for multiple primer pairs and tested on foodborne pathogens and beta-globin gene mutations.

Main Results:

  • MCPC enables detection of 2(n)-1 targets using n fluorophores in one reaction.
  • Proof-of-principle demonstrated by identifying 15 human papillomavirus types.
  • Improved MCPC with HAND system allowed detection of 10 foodborne pathogens with 10 primer pairs, achieving uniplex PCR detection limits.
  • Successfully detected combined genotypes of five beta-globin gene mutations.

Conclusions:

  • MCPC significantly expands target detection volume in real-time PCR assays.
  • The HAND system integration enhances MCPC for multiplex primer pair applications.
  • MCPC offers a powerful strategy for complex genetic analyses previously unachievable.