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Applied and Environmental Microbiology, October 1998, p. 3724-3730, Vol. 64, No. 10
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Bias in Template-to-Product Ratios in Multitemplate PCR

Martin F. Polzdagger 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.

* Corresponding author. Mailing address: The Biological Laboratories, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138. Phone: (617) 495-2177. Fax: (617) 496-5854. E-mail: ccavanaugh{at}oeb.harvard.edu.

dagger Present address: Department of Civil and Environmental Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139-4307.

Applied and Environmental Microbiology, October 1998, p. 3724-3730, Vol. 64, No. 10
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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