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Related Concept Videos

Real Time RT-PCR02:57

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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...
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An effective method based on real time fluorescence quenching for single nucleotide polymorphism detection.

Yichun Xu1, Shuai Han2, Xinhua Huang3

  • 1State Key Laboratory of Bioreactor Engineering & Shanghai Key Laboratory of Chemical Biology, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China.

Journal of Biotechnology
|July 8, 2014
PubMed
Summary

A new Quenching-PCR method rapidly detects single nucleotide polymorphisms (SNPs) using real-time fluorescence quenching. This SNP detection technology shows high accuracy, comparable to existing methods, accelerating human health research.

Keywords:
Fluorescence quenchingPCRSensitivitySingle nucleotide polymorphisms (SNPs)

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Area of Science:

  • Genetics
  • Molecular Biology
  • Biotechnology

Background:

  • Single nucleotide polymorphisms (SNPs) are common genetic variations.
  • Advancements in SNP genotyping technologies are ongoing.
  • Efficient SNP detection is crucial for understanding human health.

Purpose of the Study:

  • To introduce a novel, rapid, and effective method for SNP detection.
  • To present Quenching-PCR as an integrated real-time fluorescence quenching assay.
  • To evaluate the performance of Quenching-PCR against established methods.

Main Methods:

  • Developed Quenching-PCR, a method combining single-base extension with PCR.
  • Utilized a probe with a quencher to suppress terminal base fluorophore emission, based on dideoxy sequencing.
  • Performed real-time fluorescence detection simultaneously with dideoxy sequencing reactions.

Main Results:

  • Quenching-PCR demonstrated a high concordance rate of 93.40% compared to Sanger sequencing.
  • The TaqMan assay showed a concordance rate of 92.45%.
  • Quenching-PCR accuracy was found to be similar to the TaqMan assay.

Conclusions:

  • Quenching-PCR is a rapid and effective SNP detection technology.
  • The method offers high accuracy comparable to existing assays.
  • Quenching-PCR has potential for broad application in human health research and physiological studies.