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FIGURE 4.5 (A and B) The N2-fixing rhizobium Sinorhizobium fredii HH103, carrying the plasmid construct pMP4516 (ECFP) inside the nodule tissue of the legume host, Siratro (Stuurman et al., 2000). (C) The protozoan Tetrahymena sp. after ingesting gfp-tagged Moraxelia sp. G21 cells (Errampalli et al., 1999).

FIGURE 4.5 (A and B) The N2-fixing rhizobium Sinorhizobium fredii HH103, carrying the plasmid construct pMP4516 (ECFP) inside the nodule tissue of the legume host, Siratro (Stuurman et al., 2000). (C) The protozoan Tetrahymena sp. after ingesting gfp-tagged Moraxelia sp. G21 cells (Errampalli et al., 1999).

from soils of varying texture have been developed and these have been summarized by Bruns and Buckley (2002). There are two main approaches to nucleic acid extraction: (i) cell fractionation and (ii) direct lysis. In cell fractionation, intact microbial cells are extracted from the soil matrix. After extraction, the cells are chemically lysed and the DNA is separated from the cell wall debris and other cell contents by a series of precipitation, binding, and elution steps. In the direct lysis methods, microbial cells are lysed directly in the soil or sediment and then the nucleic acids are separated from the soil matrix by means similar to those described above. The main concerns when choosing a suitable protocol are extraction efficiency, obtaining a sample that is representative of the resident community, and obtaining an extract free of contaminants that could interfere with subsequent analyses such as PCR or hybridization with nucleic acid probes.

Extraction efficiency is a key concern for obtaining a nucleic acid extract that is representative of the soil community. Cell walls of different organisms are more or less amenable to lysis. Gram-positive organisms, resting spores and hyphae (fungi), and cysts (protozoa and nematodes) are more difficult to lyse than cells of Gram-negative organisms or vegetative stages of various soil fauna. Hence, unless lysis procedures are robust, a nucleic acid extract from soil may be biased toward organisms that are more readily ruptured. Extraction efficiency of both cell frac-tionation and direct lysis procedures can be assessed by direct microscopy, wherein extracted soil is examined for intact microbial cells using fluorescent stains. Alternatively, for assessing recovery efficiency using direct lysis procedures, soil samples may be spiked with a known quantity of labeled DNA and then the recovery of added DNA is assessed.

Obtaining a sample that is representative of the resident community is often challenging. Microbes in soil are frequently in a resting state or near starvation, making them more difficult to lyse than cells growing rapidly in culture media. Direct cell extraction protocols must ensure that cells are released from soil without bias and direct lysis procedures must ensure that nucleic acids are not adsorbed to clays or soil organic matter and thus not recovered. Failure to recover a representative sample of nucleic acids from soil is a potentially significant source of bias that may affect later data interpretation.

Coextraction of contaminants, such as humic substances, is also a common problem. Such contaminants may interfere with PCR amplification or hybridization experiments to the point where the reactions may fail entirely. Several methods have been suggested to eliminate or reduce contaminants. One approach is to use a commercial post-PCR DNA clean-up kit. While these kits are normally designed to remove salts from post-PCR amplification reactions, they may also work to reduce contaminants in DNA extracts, thereby reducing PCR inhibition in many instances. An alternative method is to subject the nucleic acid extract to an additional washing step with dilute EDTA, pass the extract through a Sephadex G-75 column, or gel purify the nucleic acid prior to analysis. In many cases, a simple dilution of the DNA extract may alleviate problems with PCR amplification, but may also dilute out soil community members of interest. It must be kept in mind that all manipulations of nucleic acid extracts can lead to loss of material and hence sparsely represented members of the community may be lost from subsequent analyses. In addition, all postextraction clean-up procedures add extra expense and processing time, thus reducing the number of samples that can be analyzed within the scope of any experiment.

Commercial soil DNA and RNA extraction kits that are based on direct lysis by bead-beating have become available. The DNA/RNA extracted is of high molecular weight and usually of sufficient quality to be used directly in PCR or nucleic acid hybridization experiments for most soils.

Many postextraction analysis procedures require that the concentration of extracted nucleic acids and their quality are known. Several methods, including fluorimetry with use of Hoechst 33258 or SYBR Green I dye, in which absorbance is recorded and compared to a calf thymus DNA standard curve, or simply gel electrophoresis against standard, commercially available, Escherichia coli DNA, can be used to estimate nucleic acid concentrations. A UV spectrophotometer allows the absorbance of the extract at 260 (DNA) and 280 nm (contaminating proteins) to be measured. The ratio between these two readings is an indicator of the purity (quality) of the DNA extract.

choosing between dna and rna for soil ecology studies

A key decision a researcher must make prior to molecular analysis of soil microbial communities is whether to extract microbial DNA, RNA, or both types of nucleic acids. DNA analysis has been used most frequently because DNA is more stable and easier and less costly to extract from soil. Postextraction analyses are straightforward and yield considerable information about the presence of various organisms in a given sample. The key problem with DNA analysis is that it does not reflect the abundance or level of activity of different organisms in a sample. When cells die, DNA released into the soil solution is rapidly hydrolyzed by nucleases. However, DNA contained in dead cells within soil aggregates or otherwise protected from decomposition will be extracted along with that from moribund and active cells. RNA on the other hand is highly labile and often difficult to extract from soil. In practice, only rRNA can be extracted with reasonable efficiency from soil at this time. A relatively simple method for ribosome extraction from soil is given in Felske et al. (1996) and commercial extraction kits are also available. Extraction of mRNA, which could be used to examine gene expression in soil under varying conditions, has been impossible until very recently. Even now, it is fraught with difficulty as mRNA is often extremely short-lived and is frequently being transcribed and translated simultaneously (see Fig. 4.2) in prokaryotes. Many postextraction analyses require that RNA is first reverse-transcribed into cDNA and then the cDNA used in downstream analyses. The advantage of extracting and analyzing RNA is that it is generally present in high amounts only in actively metabolizing cells. As substrate becomes limiting, cell processes slow down and, in some organisms, rRNA turnover may also be slowed. Thus, analysis of RNA is more reflective of the portion of the soil microbial community that is active at the time of sampling or has recently been active.

analysis of nucleic acid extracts

DNA:DNA REASSOCIATION KINETICS

When DNA is denatured by either heating or use of a denaturant (e.g., urea), the double helix structure is lost as the two strands, held by hydrogen bonds between complementary base pairs (A:T and G:C), come apart. When the denaturant is removed or the temperature is lowered, complementary strands will reanneal (Fig. 4.6). When genome complexity is low, the time it takes for all single strands to find

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