Bias in template-to-product ratios in multitemplate PCR
Open accessAbstract
Bias introduced by the simultaneous amplification of specific genes from complex mixtures of templates remains poorly understood. To explore potential causes and the extent of bias in PCR amplification of 16S ribosomal DNAs (rDNAs), genomic DNAs of two closely and one distantly related bacterial species were mixed and amplified with universal, degenerate primers. Quantification and comparison of template and product ratios showed that there was considerable and reproducible overamplification of specific templates. Variability between replicates also contributed to the observed bias but in a comparatively minor way. Based on these initial observations, template dosage and differences in binding energies of permutations of the degenerate, universal primers were tested as two likely causes of this template-specific bias by using 16S rDNA templates modified by site-directed mutagenesis. When mixtures of mutagenized templates containing AT- and GC-rich priming sites were used, templates containing the GC-rich permutation amplified with higher efficiency, indicating that different primer binding energies may to a large extent be responsible for overamplification. In contrast, gene copy number was found to be an unlikely cause of the observed bias. Similarly, amplification from DNA extracted from a natural community to which different amounts of genomic DNA of a single bacterial species were added did not affect relative product ratios. Bias was reduced considerably by using high template concentrations, by performing fewer cycles, and by mixing replicate reaction preparations.
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Amplification of diverse multitemplate mixtures using universal primers can lead to highly unequal amplification efficiencies among products.
“Amplification of diverse multitemplate mixtures using universal primers can lead to highly unequal amplification efficiencies among products [39,49].”
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- Authors
- Polz, M. F., & Cavanaugh, C. M.
- Affiliations
- Harvard University (US)
- Venue
- Applied and Environmental Microbiology
- Publisher
- American Society for Microbiology
- Published
- 1998-10
- Volume
- 64
- Issue
- 10
- Pages
- 3724-3730
- ISSN
- 0099-2240 (ISSN-L)
- 1098-5336
- PMC
- PMC106531
- OpenAlex
- W2162578061
- Cited by
- 1,484
- References
- 30
- Citations by year
- 2026: 28 · 2025: 29 · 2024: 39 · 2023: 34 · 2022: 37 · 2021: 56 · 2020: 57 · 2019: 62 · 2018: 93 · 2017: 47 · 2016: 58 · 2015: 80 · 2014: 77 · 2013: 63 · 2012: 90
- FWCI
- 7.88
- Type
- journal article
- Language
- en
- Open access
- green
- europepmc.org
- Locations
- Applied and Environmental Microbiology (publishedVersion)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Digital Access to Scholarship at Harvard (DASH) (Harvard University) (submittedVersion, cc-by)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
- Physical Sciences › Environmental Science › Ecology › Microbial Community Ecology and Physiology
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
- Keywords
- PCR bias, 16S rRNA gene, degenerate primers, gene copy number, site-directed mutagenesis, PCR cycle number, template concentration
- MeSH terms
- Bacillus subtilis, Base Sequence, DNA, Ribosomal, Escherichia coli, RNA, Ribosomal, 16S, Sensitivity and Specificity, Sequence Homology, Nucleic Acid, Templates, Genetic, Vibrio, Reproducibility of Results, Oligonucleotide Probes, Polymerase Chain Reaction, Mutagenesis, Sequence Alignment, DNA Primers
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