Lawrence Berkeley National Laboratory

Many environmental sites (e.g., groundwater, seawater, contaminated sites) contain low microbial biomass, which can make it difficult to effectively analyze these samples using genomic techniques requiring large amounts of DNA. Functional gene microarrays, for example, require microgram quantities of DNA. Whole community genome amplification (WCGA) amplifies the entire community in a sample using random priming. As such, the use of WCGA would be beneficial for samples containing low concentrations of DNA or that would be difficult to resample. Two DNA polymerases are known to catalyze this reaction: phi29 and Bst. Phi29 has been previously shown to be effective for amplifying microbial community DNA with a low amplification bias. This study evaluated the use of Bst for WCGA. Initial experiments compared the amplification of Desulfovibrio vulgaris using phi29 and Bst. An aliquot of DNA (10 ng) was amplified using random heptamers containing two additional 5-nitroindole residues on the 5' end and a phosphorothioate linkage on the 3' end. Amplified DNA (1 mu g) and the same amount of unamplified DNA were labeled with Cy5-dUTP and Cy3-dUTP, respectively. The unamplified DNA was pooled and then co-hybridized with the amplified DNA to a D. vulgaris microarray at 45 oC for 10 h. All hybridizations were done in triplicate. The signal-to-noise ratio (SNR) was calculated for the unamplified sample and all genes with SNR <3 in any of the triplicates was removed and the remaining genes were used for analysis. The average amplified to unamplified signal intensity was 1.02 (+0.005) and 1.05 (+0.005) for Bst and phi29, respectively. The representational bias (DjTotal) values were similar for both the Bst and phi29 amplifications (0.181 and 0.157, respectively). The percentage of genes whose ratios were > 1.5, 2, 3, and 4 were 12.56, 2.79, 0.15, and 0.03percent, respectively, for Bst and 12.22, 3.08, 0.23, and 0.03percent, respectively, for phi29. These results suggest that Bst would be a viable substitute for phi29. Further experiments are underway to compare the amplification of other pure culture DNA and examine microbial community DNA amplification.