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Related Concept Videos

PCR01:32

PCR

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RACE - Rapid Amplification of cDNA Ends02:35

RACE - Rapid Amplification of cDNA Ends

Rapid Amplification of cDNA Ends, or RACE, is one of the most effective methods to obtain a full-length cDNA from an mRNA sequence between a known internal region to the unknown sequence at the 5’ or 3’ end. The unknown region is cloned in the cDNA by a gene-specific primer that binds the known end, and a hybrid primer that attaches a predefined anchor sequence to the unknown end of the cDNA. The sequence in between is amplified by PCR with an anchor primer and a gene-specific primer.
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Next-generation Sequencing

The first human genome sequencing project cost $2.7 billion and was declared complete in 2003, after 15 years of international cooperation and collaboration between several research teams and funding agencies. Today, with the advent of next-generation sequencing technologies, the cost and time of sequencing a human genome have dropped over 100 fold.
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Updated: May 29, 2026

Primer Extension Capture: Targeted Sequence Retrieval from Heavily Degraded DNA Sources
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Barcoded primers used in multiplex amplicon pyrosequencing bias amplification.

David Berry1, Karim Ben Mahfoudh, Michael Wagner

  • 1Department of Microbial Ecology, Vienna Ecology Center, Faculty of Life Sciences, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria.

Applied and Environmental Microbiology
|September 6, 2011
PubMed
Summary

Multiplex amplicon sequencing with barcode-tagged PCR primers introduces bias, affecting data accuracy. A novel two-step PCR method enhances reproducibility and recovers greater genetic diversity in environmental DNA sequencing.

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Area of Science:

  • Molecular Biology
  • Environmental DNA (eDNA) Analysis
  • Bioinformatics

Background:

  • Multiplex amplicon sequencing is widely used for analyzing environmental DNA (eDNA).
  • The use of "barcode-tagged" PCR primers can introduce an amplification bias.
  • This bias leads to variability in downstream data, impacting Terminal Restriction Fragment Length Polymorphism (T-RFLP) and pyrosequencing results.

Purpose of the Study:

  • To identify and address the amplification bias associated with barcode-tagged PCR primers in multiplex sequencing.
  • To develop a more reproducible method for eDNA analysis.
  • To improve the recovery of genetic diversity in pyrosequencing libraries.

Main Methods:

  • Investigated the amplification bias introduced by "barcode-tagged" PCR primers in multiplex amplicon sequencing.
  • Developed and tested a simple two-step PCR approach.
  • Compared the reproducibility and genetic diversity recovery of the new method against standard methods using environmental DNA templates.

Main Results:

  • Identified an overlooked amplification bias inherent in "barcode-tagged" PCR primers.
  • Demonstrated that the two-step PCR approach significantly increases reproducibility.
  • Showed consistent recovery of higher genetic diversity in pyrosequencing libraries using the proposed method.

Conclusions:

  • The "barcode-tagged" PCR primer approach for multiplex sequencing introduces a significant bias affecting data reliability.
  • A simple two-step PCR protocol can overcome this bias, leading to more reproducible and accurate eDNA analyses.
  • This improved methodology enhances the assessment of genetic diversity from environmental samples.