No other area of soil ecology has developed more rapidly in recent years than the use of molecular methods to characterize the soil microbial community. The ability to extract deoxyribonucleic acids (DNA) and ribonucleic acids (RNA) from cells contained within soil samples and their direct analysis in hybridization experiments or use in polymerase chain reaction (PCR) amplification experiments have allowed us to detect and begin to characterize a vast diversity of microbes unimagined previously. Direct microscopic counts of soil bacteria are typically one to two orders of magnitude higher than counts obtained by culturing (Chap. 5). Molecular methods have the potential to provide access to this, as yet undescribed, 90-99% of the soil community.
Molecular microbial ecology relies on extracting and characterizing nucleic acids and other subcellular components, such as phospholipid fatty acids (Chap. 3), from soil organisms. Once extracted, nucleic acids or other marker molecules may be analyzed directly; or for DNA, specific target sequences may be amplified by PCR and the resulting PCR products characterized further. In the case of RNA (ribosomal (rRNA) or messenger (mRNA)), complementary DNA (cDNA) is derived from the RNA extract by reverse transcriptase (RT) PCR and the cDNA produced is analyzed subsequently. Both extracting nucleic acids from soil and amplifying them by PCR may have considerable biases associated with them and these must be taken into account when interpreting the results of subsequent analyses.
The aim of many molecular community analyses is to describe population diversity by calculating taxon richness and evenness. Due to bias in DNA and RNA extraction and PCR amplification, it is difficult, if not impossible, to assess the true abundance of different taxa using these approaches. In addition, these methods alone, although very powerful, cannot be used to assign function unambiguously to different taxa. Hence, molecular methods should be used in concert with other approaches (termed a polyphasic or multiphasic approach) to achieve a more holistic understanding of the structure and function of soil microbial communities. The focus of this chapter is on methods for extracting and analyzing soil- and sediment-derived nucleic acids and drawing ecological information from analysis results.
types and structures of nucleic acids
There are two types of nucleic acids present in all cells: DNA and RNA. These are the target molecules for most molecular analyses. The structure of DNA was deduced by Watson and Crick in 1953. They described a double helix of nucleotide bases that could "unzip" to make copies of itself. DNA was known to contain equimolar ratios of adenine (A) and thymine (T) and of cytosine (C) and guanine (G). Watson recognized that the adenine-thymine pair, held together by two hydrogen bonds, and the cytosine-guanine pair, held together by three hydrogen bonds, resulted in similar shapes and could fit together to form the rungs of a ladder of nucleotides. Molecular methods employing nucleic acids take advantage of the base-pairing rules between these four nucleotides (Fig. 4.1).
The DNA backbone is composed of deoxyribose (sugar), phosphates, and the associated purine (A and G) and pyrimidine (T and C) bases. It is the base-pairing specificity between the nucleotides that leads directly to the faithful copying of both strands of the DNA double helix during replication and that can be exploited to make copies of selected genes (or regions of the DNA molecule) in vitro by use of the PCR.
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