DNA extraction from low-biomass carbonate rock: An improved method with reduced contamination and the low-biomass contaminant database

Caves represent a unique environment in which to study subsurface geomicrobial interactions and processes. One of the primary techniques used to study such geologic samples is molecular phylogenetic analysis, but this technique is hampered by low microbial biomass and calcium in the host rock, often leading to poor and irreproducible DNA extraction. We describe an improved protocol to recover extremely low amounts of DNA from calcium-rich geologic samples. This protocol relies on the use of the synthetic DNA molecule poly-dIdC, to act both as blocking agent and carrier molecule to increase the yield of DNA, and dialysis to remove calcium inhibitors of PCR amplification. Further, we demonstrate that many traditionally used laboratory substrates contain microbial DNA that can be amplified through the polymerase chain reaction (PCR) and contaminate molecular phylogenetic profiles. While the number of potential contaminants can be minimized, it cannot be eliminated from extraction techniques. We have therefore established the low-biomass contaminant (LBC) database, which contains the 16S rRNA gene sequences of species that have been identified as common laboratory contaminants. These identified contaminants provide a reference database to allow investigators to critically evaluate certain species identified within their phylogenetic profile when examining such low-biomass environments.