CiteULike: vrich's Cavanaugh

Intragenomic Variation and Evolution of the Internal Transcribed Spacer of the rRNA Operon in Bacteria

Stewart — 2007-11-14T09:59:10-00:00

Journal of Molecular Evolution, Vol. 65, No. 1. (July 2007), pp. 44-67.

Bias in Template-to-Product Ratios in Multitemplate PCR

Martin Polz — 2008-05-04T21:02:13-00:00

Appl. Environ. Microbiol., Vol. 64, No. 10. (1 October 1998), pp. 3724-3730.

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.

Diversity and Heterogeneity of Epibiotic Bacterial Communities on the Marine Nematode Eubostrichus dianae

Martin Polz — 2008-05-04T19:59:30-00:00

Appl. Environ. Microbiol., Vol. 65, No. 9. (1 September 1999), pp. 4271-4275.

The diversity of a microbial community covering the surface of a marine nematode was analyzed by performing a 16S ribosomal DNA (rDNA) restriction cutting and sequencing analysis. In two clone libraries constructed by using individual nematodes, 54 and 85 restriction patterns were identified, and only 13 of these patterns were common to both libraries. Sequence analysis indicated that the common patterns belonged to four groups related to sequences of cytophagas, sulfate-reducing bacteria, members of the gamma subclass of the class Proteobacteria, and caulobacters. At least two groups appeared to be permanent members of the community as they were also detected in a 16S rDNA library constructed 3 years previously by using 100 pooled nematode specimens. A surprising outcome was that very dominant filamentous bacteria were apparently not represented in the clone libraries, as quantitative probing showed that none of the common operational taxonomic unit groups displayed the expected overwhelming dominance. Nevertheless, our analysis revealed both an unexpectedly high level of bacterial diversity and heterogeneity in samples representing presumably very similar microenvironments.