Bias in Template-to-Product Ratios in Multitemplate PCR

ABSTRACT 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.

[1]  J. Saunders,et al.  Microbial Evolution, Diversity, and Ecology: A Decade of Ribosomal RNA Analysis of Uncultivated Microorganisms , 1998, Microbial Ecology.

[2]  L. Kerkhof,et al.  Ribosomal RNA gene dosage in marine bacteria. , 1997, Molecular marine biology and biotechnology.

[3]  F. Brockman,et al.  Effect of PCR template concentration on the composition and distribution of total community 16S rDNA clone libraries , 1997, Molecular ecology.

[4]  M. Polz,et al.  A simple method for quantification of uncultured microorganisms in the environment based on in vitro transcription of 16S rRNA , 1997, Applied and environmental microbiology.

[5]  D. Stahl,et al.  Characterization of universal small-subunit rRNA hybridization probes for quantitative molecular microbial ecology studies , 1996, Applied and environmental microbiology.

[6]  D. Bezdicek,et al.  Estimation of the abundance of an uncultured soil bacterial strain by a competitive quantitative PCR method , 1996, Applied and environmental microbiology.

[7]  Daniel B. Oerther,et al.  The oligonucleotide probe database , 1996, Applied and environmental microbiology.

[8]  S. Giovannoni,et al.  Bias caused by template annealing in the amplification of mixtures of 16S rRNA genes by PCR , 1996, Applied and environmental microbiology.

[9]  Ross A. Overbeek,et al.  The Ribosomal Database Project (RDP) , 1996, Nucleic Acids Res..

[10]  M. Polz,et al.  Phylogenetic relationships of the filamentous sulfur bacterium Thiothrix ramosa based on 16S rRNA sequence analysis. , 1996, International journal of systematic bacteriology.

[11]  E. Stackebrandt,et al.  Effect of genome size and rrn gene copy number on PCR amplification of 16S rRNA genes from a mixture of bacterial species , 1995, Applied and environmental microbiology.

[12]  C. Hopkinson,et al.  Production and utilization of dissolved organic carbon during an experimental diatom bloom , 1995 .

[13]  W. J. Lucas,et al.  In situ isolation of mRNA from individual plant cells: creation of cell-specific cDNA libraries. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[14]  G. Mutter,et al.  PCR bias in amplification of androgen receptor alleles, a trinucleotide repeat marker used in clonality studies. , 1995, Nucleic acids research.

[15]  K. Schleifer,et al.  Phylogenetic identification and in situ detection of individual microbial cells without cultivation. , 1995, Microbiological reviews.

[16]  J. W. Pendleton,et al.  Surveys of Gene Families Using Polymerase Chain Reaction: PCR Selection and PCR Drift , 1994 .

[17]  D. Stahl,et al.  Quantification of methanogenic groups in anaerobic biological reactors by oligonucleotide probe hybridization , 1994, Applied and environmental microbiology.

[18]  D. Stahl,et al.  Group-specific 16S rRNA hybridization probes to describe natural communities of methanogens , 1994, Applied and environmental microbiology.

[19]  A. Uitterlinden,et al.  Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA , 1993, Applied and environmental microbiology.

[20]  A. Sardelli.  Plateau effect-understanding PCR limitations , 1993 .

[21]  N. Pace,et al.  Differential amplification of rRNA genes by polymerase chain reaction , 1992, Applied and environmental microbiology.

[22]  K. Jarrell,et al.  A general method of isolating high molecular weight DNA from methanogenic archaea (archaebacteria). , 1992, Canadian journal of microbiology.

[23]  N. Arnheim,et al.  Polymerase chain reaction strategy. , 1992, Annual review of biochemistry.

[24]  J. Sninsky,et al.  Recent advances in the polymerase chain reaction , 1991, Science.

[25]  D. Lane.  16S/23S rRNA sequencing , 1991 .

[26]  E. Stackebrandt,et al.  Nucleic acid techniques in bacterial systematics , 1991 .

[27]  R. Atlas,et al.  Polymerase chain reaction: applications in environmental microbiology. , 1991, Annual Review of Microbiology.

[28]  V. Torsvik,et al.  High diversity in DNA of soil bacteria , 1990, Applied and environmental microbiology.

[29]  B Flesher,et al.  Use of phylogenetically based hybridization probes for studies of ruminal microbial ecology , 1988, Applied and environmental microbiology.

[30]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .