Size of an actual DNA microarray
FIGURE 4.7 Steps in developing and using a microarray. (A) Flow chart depicting the steps from extracting mRNA from a sample to reading the microarray with a laser scanner (Campbell and Reece, 2005). (B) Detail of how the labeled cDNA interacts with the probes bound to the microarray and how a positive signal from one or the other sample is detected. (C) An image of a microarray reading (http://www.bsi.vt.edu/ralscher/gridit/).
using this approach. Techniques to improve the sensitivity have been reported by Denef et al. (2003). Nonspecific binding of target nucleic acids to the probes is also a serious issue that needs to be overcome (Zhou and Thompson, 2002). The details of microarray construction and types of arrays can be found in Ekins and Chu (1999) and ecological applications are reviewed in Zhou (2003).
There are three basic types of arrays used in soil ecology: (i) community genome arrays (CGA), used to compare the genomes of specific groups of organisms; (ii) functional gene arrays (FGA), used to detect the presence of genes of known function in microbial populations in prepared soil samples and more recently used to detect gene expression; and (iii) phylogenetic oligonucleotide arrays (POA), used to characterize the relative diversity of organisms in a sample through the use of rRNA sequence-based probes. Whole genomic DNA, requiring culturing of target organisms, is used to develop the probes used in CGA. Both oligonucleotides and DNA fragments derived from functional genes, such as those involved in C, N, S, and metal cycling, can be used to prepare FGAs and query the status of these functional genes within a soil community. In POA, both conserved- and variable-region rRNA gene sequences are used to determine the presence of particular phylogenetic groups in a given sample.
RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP) ANALYSIS
In RFLP analysis, total DNA purified from soil is hydrolyzed with a restriction endonuclease (often EcoRI or HindIII). Restriction enzymes, produced by and originally isolated from various bacteria, cut double-stranded DNA at palindromic sequences (those that read in the same order both backward and forward). Restriction enzymes can be selected that cut either frequently or infrequently along the isolated DNA strands. The variation (polymorphisms) in the length of resulting DNA fragments is visualized by running the DNA fragments on an electrophoretic gel and staining the gel with ethidium bromide or SYBR Green I, which fluoresce under UV light. These variations in fragment lengths are then used as a "fingerprint," to differentiate between soil communities. Discrimination between communities is often difficult because of the large number of fragments generated and the difficulty in resolving closely spaced bands in a gel. RFLP is rarely used on its own for diversity studies. It is used most commonly in conjunction with the Southern method of transferring (blotting) the DNA fragments from the gel onto a nitrocellulose or other membrane and then probing the blot with appropriately labeled oligonucleotide gene probes to test for the presence of specific sequences or used in conjunction with PCR of amplified ribosomal genes in a technique called ARDRA (amplified ribosomal DNA restriction analysis). The ultimate aim of RFLP-based methods is to be able to compare differences between DNA fingerprints obtained from different communities. Observed differences can then be characterized more fully using other methods, largely involving cloning and sequencing of DNA fragments of interest.
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