EFFECT OF PCR TEMPLATE CONCENTRATION ON THE COMPOSITION AND DISTRIBUTION OF TOTAL COMMUNITY 16S RDNA CLONE LIBRARIES

Total DNA from sediment samples was isolated by a direct lysis techniq ue. Purified DNA was used as template either undiluted or diluted 1 : 10 prior to polymerase chain reaction (PCR) amplification of 16S rRNA genes. Full-length inserts were analysed for restriction fragment leng th polymorphisms (RFLP) with the enzyme Cfo1, and the resulting distri bution and abundance of RFLP patterns compared between the undiluted a nd diluted PCR reactions. Results indicate that for low PCR template c oncentrations, in the range from a few picograms to tens of picograms DNA, proportional representation of specific RFLP types was not reprod ucible upon template dilution, confirming that PCR amplification of 16 S rDNA cannot be used directly to infer microbial abundance. In partic ular, only 15-24% of the RFLP types recovered from a sample were prese nt in both the undiluted and diluted extracts. We propose that very lo w template concentrations in the PCR generate random fluctuations in p riming efficiency, which led to the contrast in the RFLP types observe d in the libraries from the undiluted and diluted extracts.