The study investigates the bias in template-to-product ratios during PCR amplification of 16S ribosomal DNAs (rDNAs) from complex mixtures of templates. The researchers used genomic DNAs from two closely related and one distantly related bacterial species, amplified them with universal, degenerate primers, and quantified and compared the template and product ratios. They found significant and reproducible overamplification of specific templates, with variability between replicates contributing to observed bias. The study tested two potential causes of this bias: template dosage and differences in binding energies of degenerate primers. Using 16S rDNA templates modified by site-directed mutagenesis, they found that GC-rich permutations of primers amplified templates more efficiently, indicating that primer binding energies are a major factor in overamplification. However, gene copy number was not found to be a significant cause of bias. The study also showed that reducing the number of PCR cycles and using high template concentrations can reduce bias. Overall, the results suggest that PCR analysis has high precision but low accuracy due to template-inherent factors, and provide recommendations to minimize bias in PCR amplifications.The study investigates the bias in template-to-product ratios during PCR amplification of 16S ribosomal DNAs (rDNAs) from complex mixtures of templates. The researchers used genomic DNAs from two closely related and one distantly related bacterial species, amplified them with universal, degenerate primers, and quantified and compared the template and product ratios. They found significant and reproducible overamplification of specific templates, with variability between replicates contributing to observed bias. The study tested two potential causes of this bias: template dosage and differences in binding energies of degenerate primers. Using 16S rDNA templates modified by site-directed mutagenesis, they found that GC-rich permutations of primers amplified templates more efficiently, indicating that primer binding energies are a major factor in overamplification. However, gene copy number was not found to be a significant cause of bias. The study also showed that reducing the number of PCR cycles and using high template concentrations can reduce bias. Overall, the results suggest that PCR analysis has high precision but low accuracy due to template-inherent factors, and provide recommendations to minimize bias in PCR amplifications.