Comparisons of different hypervariable regions of rrs genes for use in fingerprinting of microbial communities by PCR-denaturing gradient gel electrophoresis

Abstract

Denaturing gradient gel electrophoresis (DGGE) has become a widely used tool to examine microbial diversity and community structure, but no systematic comparison has been made of the DGGE profiles obtained when different hypervariable (V) regions are amplified from the same community DNA samples. We report here a study to make such comparisons and establish a preferred choice of V region(s) to examine by DGGE, when community DNA extracted from samples of digesta is used. When the members of the phylogenetically representative set of 218 rrs genes archived in the RDP II database were compared, the V1 region was found to be the most variable, followed by the V9 and V3 regions. The temperature of the lowest-melting-temperature (T(m(L))) domain for each V region was also calculated for these rrs genes, and the V1 to V4 region was found to be most heterogeneous with respect to T(m(L)). The average T(m(L)) values and their standard deviations for each V region were then used to devise the denaturing gradients suitable for separating 95% of all the sequences, and the PCR-DGGE profiles produced from the same community DNA samples with these conditions were compared. The resulting DGGE profiles were substantially different in terms of the number, resolution, and relative intensity of the amplification products. The DGGE profiles of the V3 region were best, and the V3 to V5 and V6 to V8 regions produced better DGGE profiles than did other multiple V-region amplicons. Introduction of degenerate bases in the primers used to amplify the V1 or V3 region alone did not improve DGGE banding profiles. Our results show that DGGE analysis of gastrointestinal microbiomes is best accomplished by the amplification of either the V3 or V1 region of rrs genes, but if a longer amplification product is desired, then the V3 to V5 or V6 to V8 region should be targeted.

FIG. 1.

Frequency distribution of T_m(L) values for the V1 region (A), V3 region (B), and V5 region (C), derived from the phylogenetically representative set of 218 rrs gene sequences archived in RDP II (release 8.1). The arrows indicate the average T_m(L) value calculated for each V region, and the T_m(L) values appear to be normally distributed.

FIG. 2.

DGGE banding profiles of V regions produced from the community DNA extracted from eight different samples of ruminal digesta. The nondegenerate primers and DGGE conditions described in Table 1 were used in the PCR and DGGE. The lanes labeled C1 and C2 represent samples of digesta collected from animals fed a mixture of hay and grain, and the lanes labeled F1 and F2 represent samples of digesta collected from animals fed a mixture of hay, grain, and beef tallow. D14 and D23, samples collected 14 and 23 days, respectively, after the animals were started on the diet; Marker, electrophoresis marker; V6 to V8^a, DGGE profiles generated using primers GC-954f and 1369r; V6 to V8^b, DGGE profiles generated using primers F-968-GC and R-1401.

FIG. 3.

DGGE gel banding profiles of the V1 and V3 regions produced with V1-specific inosine-containing primers (A), V3-specific degenerate primers (B), or V3-specific inosine-containing primers (C). The labeling of lanes follows the same protocol as described for Fig. 2.