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Related Concept Videos

PCR01:32

PCR

Overview
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...
PCR - Polymerase Chain Reaction01:32

PCR - Polymerase Chain Reaction

Overview

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Primers with 5' flaps improve real-time PCR.

Irina Afonina1, Irina Ankoudinova, Alan Mills

  • 1Nanogen Inc., Bothell, WA 98021, USA. iafonina@nanogen.com

Biotechniques
|February 7, 2008
PubMed
Summary

Short 5

Area of Science:

  • Molecular Biology
  • Biochemistry

Background:

  • Primers with noncomplementary sequences are used to add utility sequences to PCR products.
  • Optimizing PCR amplification and fluorescent signal detection is crucial for molecular diagnostics.

Purpose of the Study:

  • To investigate the effect of primer 5' overhangs on real-time PCR fluorescent signal.
  • To determine if AT-rich overhangs enhance amplification of challenging DNA templates.

Main Methods:

  • Synthesized primers with varying 5' overhang sequences.
  • Performed real-time PCR using these primers on standard and difficult-to-amplify DNA templates.
  • Measured and compared fluorescent signals generated during amplification.

Main Results:

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  • Primers with short 5' AT-rich overhangs significantly increased real-time PCR fluorescent signal.
  • This enhancement was particularly notable for difficult-to-amplify templates like viral sequences and bisulfite-treated DNA.
  • The AT-rich overhangs improved the efficiency of PCR amplification.

    • Conclusions:

    • Incorporating short 5' AT-rich overhangs in primers is an effective strategy to boost real-time PCR fluorescent signals.
    • This method offers a simple yet powerful approach to improve the detection of low-abundance or complex nucleic acid targets.
    • The findings have implications for enhancing sensitivity in molecular assays and diagnostics.