Bias in Template-to-Product Ratios in Multitemplate PCR Martin F. Polz and Colleen M. Cavanaugh The Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138 Received 13 April 1998/Accepted 13 July 1998 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. PCR has become an invaluable tool because of the speed and simplicity with which specific DNA segments can be amplified from a background of complex genomes. In studies of molecular evolution and microbial ecology, this property has facilitated the characterization of both single genes and families of related genes in single or multiple species. This is generally done by designing degenerate primers which target conserved regions of homologous genes, thereby accelerating the detection, amplification, and, ultimately, sequence analysis of the genes under study. One of the most innovative applications of the PCR has been the cataloging of bacterial and archaeal species richness in the environment. Mixtures of 16S rRNA genes amplified from natural communities are considered representative of the native organisms from which they originated. This approach has revealed the existence of numerous uncultured microorganisms because it circumvents bias introduced by traditional culture-based methods. The protocols involve extraction of nucleic acids from an environmental sample, PCR amplification of the 16S rRNA genes with universal, degenerate primers, and separation of amplified products by cloning or by denaturing gradient gel electrophoresis (DGGE). Subsequently, clones or bands on DGGE gels canBias in Template-to-Product Ratios in Multitemplate PCR Martin F. Polz and Colleen M. Cavanaugh The Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138 Received 13 April 1998/Accepted 13 July 1998 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. PCR has become an invaluable tool because of the speed and simplicity with which specific DNA segments can be amplified from a background of complex genomes. In studies of molecular evolution and microbial ecology, this property has facilitated the characterization of both single genes and families of related genes in single or multiple species. This is generally done by designing degenerate primers which target conserved regions of homologous genes, thereby accelerating the detection, amplification, and, ultimately, sequence analysis of the genes under study. One of the most innovative applications of the PCR has been the cataloging of bacterial and archaeal species richness in the environment. Mixtures of 16S rRNA genes amplified from natural communities are considered representative of the native organisms from which they originated. This approach has revealed the existence of numerous uncultured microorganisms because it circumvents bias introduced by traditional culture-based methods. The protocols involve extraction of nucleic acids from an environmental sample, PCR amplification of the 16S rRNA genes with universal, degenerate primers, and separation of amplified products by cloning or by denaturing gradient gel electrophoresis (DGGE). Subsequently, clones or bands on DGGE gels can