Pcr Fingerprinting

PCR fingerprinting can be accomplished by several different methods, all of which are aimed at distinguishing differences in the genetic makeup of microbial populations from different samples. The advantage of these techniques is that they are rapid in comparison with sequencing methods, thus enabling high sample throughput, and can be used to target sequences that are phylogenetically or functionally significant. Depending on the primers chosen, PCR fingerprints can be used to distinguish between isolates at the strain level or to characterize target microbes at the community level. The more common PCR fingerprinting techniques in use today for characterizing soil microbial community composition are DGGE or TGGE (Muyzer and Smalla, 1998) and terminal restriction fragment length polymorphism (T-RFLP) analysis (Liu et al., 1997; Clement et al., 1998). Both techniques can be used to separate PCR products that are initially of a similar length by employing additional methods to separate the amplicons into a greater number of bands that are then used for community comparisons.

DGGE and TGGE are identical in principle (Fig. 4.10). Both techniques impose a parallel gradient of denaturing conditions along a polyacrylamide gel. Double-stranded DNA (dsDNA) PCR amplicons are loaded in wells at the top of the gel and, as the DNA migrates, the denaturing conditions of the gel gradually increase. In DGGE, the denaturant is typically urea; in TGGE it is temperature. Because native dsDNA is a compact structure, it migrates faster than partially denatured DNA. The sequence of a fragment determines the point in the gradient gel at which denaturation will start to retard mobility. Sequence affects duplex stability by both percentage G + C content and neighboring nucleotide interactions (e.g., GGA is more stable than GAG). The resulting gel yields a ladder of bands in each lane characteristic of the DNA extracted and amplified from the original sample. There is not a direct correspondence between bands in the DGGE gel and organism diversity, however. Sequences amplified from the DNA of different organisms may have similar melting properties in the presence of the denaturant and thus occupy the same band in the denaturing gel. DNA fragments cloned from different bands may yield as many different sequences as clones analyzed. Since there is not a one-to-one correspondence between bands and taxa, the bands are referred to as operational taxonomic units (OTUs). The OTUs form the basis of similarity and multivariate analyses of data derived from various soil communities.

While the power of DGGE and TGGE to detect PCR amplicon diversity within a single gel is high, the sensitivity of the technique to variations in experimental conditions makes comparisons between gels very difficult. These techniques are therefore of greatest use in a preliminary screening to aid recognition of sample diversity. The resolving power of these and other gel-based analyses is limited by the number of bands capable of "fitting" and being counted as individual bands on a single gel. In practice, no more than 100 distinct sequence types may be resolved despite the potential for single base-pair sensitivity. An important advantage that DGGE analysis has over T-RFLP (see below) is that PCR amplicons of interest that are resolved on a DGGE gel can be excised from the gel, reamplified, cloned, and sequenced, thereby obtaining taxonomic and/or phylogenetic information about amplifiable members of the soil community. For phylogenetic assignment of cloned sequences, variable regions within the SSU rRNA genes are amplified. An important

FIGURE 4. lO (A) Schematic representation of the denaturing gradient gel electrophoresis (DGGE) technique. In DGGE. the forward primer if318) is tagged with a GC clamp to prevent the double strands from completely separating. Along with the reverse primer (r518), it amplifies the V3 region of the 16S rRNA gene in target DNA. (B) When amplified DNA products are loaded on a denaturing gradient gel and ran in an electric field, the products separate according to their total ratio of A:T vs G:C base pairs and the locations of these base pairs relative to each other (with permission from R. Kantety. Alabama A&M).

FIGURE 4. lO (A) Schematic representation of the denaturing gradient gel electrophoresis (DGGE) technique. In DGGE. the forward primer if318) is tagged with a GC clamp to prevent the double strands from completely separating. Along with the reverse primer (r518), it amplifies the V3 region of the 16S rRNA gene in target DNA. (B) When amplified DNA products are loaded on a denaturing gradient gel and ran in an electric field, the products separate according to their total ratio of A:T vs G:C base pairs and the locations of these base pairs relative to each other (with permission from R. Kantety. Alabama A&M).

disadvantage of the gradient gel approach is that the amplicon size must be restricted to under 600 bp in length to optimize separation within the gel matrix. Therefore, full-length rRNA gene sequences cannot be recovered using these methods. DGGE and TGGE are now being applied frequently in soil microbial ecology to compare the structures of complex microbial communities and to study their dynamics.

T-RELP analysis, as in DGGE analysis, begins with amplifying soil community DNA using targeted primers, but with the key differences that one or both primers are labeled with a fluorochrome(s) and that resulting amplicons are cut with restriction enzymes to create DNA fragments of varying size, fluor-labeled at either the 5' or the 3' end (Fig. 4.11). These terminal fragments are then sized against

(A) Forward primer

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