Culture-independent molecular techniques for soil microbial ecology

The advent of nucleic acid-based molecular methods, in particular the polymerase chain reaction (PCR), has revolutionised the study of soil microbial ecology, previously constrained by an inability to culture the majority of cells detected by direct microscopic observation. Extraction of DNA and RNA directly from cells in soil circumvents the requirement to grow microorganisms in laboratory culture, avoiding problems associated with the differential growth rates of the estimated 1% that can be grown routinely. However, not all cells that contain DNA are capable of growth, and in some conditions such as air-dried soil, DNA can be extracted from non-viable microorganisms after 140 years of storage. To investigate the active microbial community, RNA can also be isolated directly from soil. Analysis of ribosomal RNA (rRNA) indicates the dominant active population in any particular set of conditions and the large, constantly increasing electronic database of gene sequences for the small subunit of rRNA (16S for prokaryotes, 18S for eukaryotes) provides identification of many soil bacteria, archaea and fungi with varying degrees of certainty to the genus, species or sub-species level. More precise information on which functional genes are active can be obtained from messenger RNA (mRNA). Newer methods including high-throughput (massively parallel) sequencing and microarrays offer further advances. We describe a range of molecular techniques used to investigate soil microbial communities, discuss how they relate to other methods for investigating bacterial and fungal activity, and explore their drawbacks and limitations.