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PCR

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Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies

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Published on: May 22, 2012

'Next-base' effect on PCR amplification.

Eitan Ben-Dov¹, Orr H Shapiro, Ariel Kushmaro

¹Avram and Stella Goldstein-Goren Department of Biotechnology Engineering, Ben-Gurion University of the Negev, PO Box 653, Be'er Sheva 84105, Israel Achva Academic College MP Shikmim, 79800, Israel National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, PO Box 653, Be'er-Sheva 84105, Israel School of Materials Science and Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798, Singapore.

Environmental Microbiology Reports

|June 13, 2013

Summary

This summary is machine-generated.

Variable bases near PCR primers can bias microbial community surveys. Lowering PCR extension temperature to 70°C minimizes this bias, improving the accuracy of 16S rRNA gene sequencing for microbial ecology.

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Area of Science:

Microbial Ecology
Molecular Biology
Genetics

Background:

PCR primers targeting the 16S rRNA gene can introduce bias in microbial community composition analysis.
The base adjacent to the 3' end of PCR primers is a suspected source of this bias.

Purpose of the Study:

To investigate the impact of the 3'-end base of PCR primers on amplification efficiency.
To determine optimal PCR conditions to minimize amplification bias in microbial surveys.

Main Methods:

Quantitative PCR was used to evaluate amplification efficiencies of custom templates and primers.
Primer extension temperatures were varied (70°C to 74°C).
Primer length was manipulated to alter the position of the variable base relative to the primer.

Main Results:

Templates with guanine or cytosine adjacent to the primer showed improved amplification efficiency at higher extension temperatures (72-74°C).
Lowering the extension temperature to 70°C reduced this observed bias.
Shortening primers attenuated but did not eliminate the bias.

Conclusions:

The nucleotide composition at the 3' end of PCR primers influences amplification bias.
Optimizing PCR extension temperature, specifically lowering it to 70°C, can mitigate bias in 16S rRNA gene surveys.
Primer design for microbial ecology studies should consider next-base nucleotide composition to ensure accurate representation of microbial communities.