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FIGURE 4.2 The relationship between DNA gene sequence, transcription into mRNA, and translation into the coded protein sequence (with permission from Addison Wesley Longman, Inc., 1999).

use of nucleic acid analyses for soil ecology studies

A wide range of techniques are available for nucleic acid analysis. These techniques fall into three basic categories: (i) analysis of nucleic acids in situ, (ii) direct analysis of extracted DNA/RNA, and (iii) analysis of PCR-amplified segments of the DNA molecule. In PCR-based approaches, the primers chosen dictate the target for amplification, such as rRNA genes or genes that code for proteins with functions of ecological interest (those involved in nitrogen fixation, ammonia or methane oxidation, or denitrification). Some of the techniques used more commonly and their interrelationships with each other and traditional methods of microbial analysis are shown in Fig. 4.3. Most methods require that target molecules be separated from the soil matrix prior to analysis. A few techniques, such as fluorescence in situ hybridization (FISH), do not. Much of our ability to make sense of results of molecular analyses and to design more robust methods depends on the continuing development of nucleic acid and protein sequence databases and associated bioinformatics analytical tools. Through comparative sequence analysis, targeted primers for use in PCR assays and probes for use in hybridization studies can be designed that have the required level of specificity for a given study.

Microscopic counts Viable plate counts Most probable number Microbial biomass ATP content

TAXONOMY/FUNCTION

FISH In situ PCR

Microscopic counts Viable plate counts Most probable number Microbial biomass ATP content

TAXONOMY/FUNCTION

ACTIVITY/FUNCTION

Stable isotope probing STARFISH

Respiration Decomposition N/P mineralization Soil enzymes

Respiration Decomposition N/P mineralization Soil enzymes

ACTIVITY/FUNCTION

Re-association kineticsk

Soil sample

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