T

B^ Electrophoresis

B^ Electrophoresis

FIGURE 4.11 Schematic representation of the terminal restriction fragment length polymorphism (T-RFLP) technique. In T-RFLP, nearly the entire 16S rRNA gene is targeted with the primers f27 and r1492. The forward primer is tagged with a fluorochrome to facilitate length assignment of the terminal fragment after the amplified products are cut with a restriction endonuclease. The final step is to run the enzyme-digested products on a DNA sequencer, which will detect and report the lengths of all of the fluorochrome-labeled terminal fragments (with permission from R. Kantety, Alabama A&M).

a standard molecular size marker using automated DNA sequencing techniques. The resulting electropherogram (peaks representing the sizes of the terminal restriction fragments, TRFs) is used as a DNA fingerprint characteristic of the soil community sampled. Resulting TRF sizes are analogous to bands on a DGGE gel and are also referred to as OTUs, since any one terminal fragment size is not restricted to any taxonomic group per se (Liu et al., 1997; Gruntzig et al., 2002). TRF profiles are compared subsequently between samples by use of similarity matrices and multivariate statistics.

Although first described in 1997, there are now over 600 studies employing this technique of which over 100 report its use for the analysis of soil microbial communities. Liu et al. (1997) used T-RFLP to characterize microbial diversity within bioreactor sludge, aquifer sand, and termite guts. Other studies have applied the technique to compare rhizosphere microbial communities colonizing transgenic and nontransgenic plants (Devare et al., 2004), the effects of soil management on fungal community composition (Edel-Hermann et al., 2004), and the effects of heavy metals on bacterial communities (Turpeinen et al., 2004),

TABLE 4.4 Comparison of the Number of Operational Taxonomic Units Generated by Four Different Molecular Community Analysis Methods for Each Samplea (Jones and Thies, in press)

Method

DGGE T-RFLP ARISA 2D-PAGE

Soil type Sample 1 2 12 12 12

DGGE T-RFLP ARISA 2D-PAGE

Soil type Sample 1 2 12 12 12

Low Zn

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