5. Optimization of DNA Extraction and DGGE Staining Techniques
5.2 Results and Discussion
5.2.1 Evaluation of DNA isolation methods for PCR-DGGE analysis of bacterial
5.2.1.3 Influence of DNA extraction method on bacterial community structure and
5.2.1.3 Influence of DNA extraction method on bacterial community structure and
Table 5.2 Influence of DNA isolation method on the numbr of bands visualized by Denaturing Gradient Gel Electrophoresis generated from identical soil samples.
Number of DNA Bands per Sample on Denaturing Gradient Gels DNA
Extraction
Method M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 +
Mo-Bio
Kit 12 21 22 17 18 17 16 19 21 22 18 19 19 20 16 15 11 11
Bead Beat
Method 11 12 14 11 20 19 16 14 16 17 18 23 16 21 13 12 10 10
*Samples 1-12 originated from soil array Ah that was destructively sampled after 12 weeks of leaching with the synthetic leachate
*Samples 13-15 originated from soil perfused with water only
*Sample 16 was untreated soil
*M was the DGGE marker (Vrije Universiteit van Amsterdam, H.W. van Verseveld)
*+ Bacillus soil isolate
The DGGE marker and Bacillus soil isolate (positive control) produced similar patterns in both gels but were more difficult to visualise on the gel containing the PCR products originating from Bead Beat isolated DNA due to dark background stain of the gradient gel. This contributed significantly to the inability to capture some of the bands in lanes with relatively lighter background staining as a consequence of contrast limitations on the gel documentation system. Conversely, the gel containing the PCR products originating from Kit isolated DNA developed with a uniform background stain making the capture of the image relatively uncomplicated (Plate 5.3). There was evidence of smearing for all the samples depicted on Plate 5.3 (b) and in some instances this made it difficult to distinguish between individual bands. On investigating the spatial homogeneity of bacteria in grassland soils, Felske and Akkermans (1998a) generated community profiles consisting of a mixture of high; medium; and low intensity bands. They noticed that the bands of lower intensity sometimes resulted in smears in the profiles generated and this impacted on the inability to separate individual bands of this intensity. The prominent bands for all of the samples are more evident in Plate 5.3 (a) than on Plate 5.3 (b). The darker bands appear to be more distinct on both gels, whereas the lighter bands are either absent or more difficult to visualise on Plate 5.3 (b). PCR amplified products for both gels were prepared, electrophoresed, and stained simultaneously so one can cautiously assume that variations in the quality of the gel images may be attributed to the quality and quantity of the PCR products which were the result of DNA isolated by two different methods. Gelsomino et al. (1999) noticed a similar trend when they compared direct and indirect DNA isolation methods on a Flevo silt loam soil from Waginingen in the Netherlands.
Plate 5.3 Silver stained Denaturing Gradient Gels illustrating the community profiles generated for common soil samples (Lanes 2 – 16) exposed to; (a) Mo-Bio Ultra Clean Soil DNA Isolation Kit and (b) Modified version of Duarte et al.(1998) Bead Beat DNA Isolation method. Lanes M and + correspond to the DGGE Marker and a Bacillus isolate from soil. The arrow (←) represents distinct bands visible on both gels.
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 + (a)
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 + (b)
Cluster analysis performed on the DGGE profiles using the unweighted pairwise grouping method with mathematical averages (UPGMA, Dice co-efficient of similarity) showed dissimilar cluster relationships between samples for each DNA isolation method (Figure 5.2). For example, PCR-DGGE of samples isolated using the Kit method showed clustering for samples 3 and 4 at 50% similarity with sample 2 joining the cluster directly at 40%. PCR-DGGE of the bead-beat isolated DNA showed that samples 3 and 4 also clustered at 50% but sample 2 only clustered indirectly at 10% similarity. Samples 5/6/7 formed a discrete cluster at 60% for the Kit isolated DNA products, with samples 5 and 6 clustering at 60% for the Bead Beat method and only indirectly pairing with sample 7 at 10%
correspondence. Samples 8 and 9 for the Kit isolated products clustered at 60% while samples 10/11/12/13 paired as a discrete cluster at 65%. Both of these distinct clusters paired at 40%.
The Bead Beat method paired samples 8 and 9 at 50%, with 10/11/13 pairing at 40% as a discrete cluster. However sample 12 joined both clusters indirectly at only 15% similarity.
The controls leached with water both formed discrete clusters at 35% and 30% for the products of Kit and Bead Beat isolation respectively. However, the phylogenetic tree for the Kit isolated DNA products was rooted by the original soil sample (17) which paired indirectly with cluster 14/15/16 at 15% whereas pairing for the Bead Beat method occurred at double the similarity to samples 15 and 16 only. For this method sample 17 served as a link between samples 15/16 and with sample 14 thereby linking cluster 15/16/17 and rooting this tree as the out-group for all the samples. This original soil sample was linked to all the samples of the investigation at 15% for the Kit isolated PCR products while pairing for the Bead Beat method occurred below 10% similarity.
The divergent phylogenetic relationships between the samples for each method relates to the presence/absence of DNA bands on the gels. The results suggest that some degree of similarity exists between the DNA isolation methods by virtue of similar clustering relationships between samples. However there are undoubtedly major differences in the number of bands depicted by PCR-DGGE as well as the prominence of specific bands isolated by each method. In this case the two DNA isolation techniques produced different phylogenetic relationships for identical soil samples. Other researchers have reported similar conclusions (Niemi et al., 2001; Lipthay et al., 2004). Lipthay et al. (2004) further concluded
that the choice of DNA isolation protocol must also impact on the functional diversity of the profiled community.
Figure 5.2 Cluster analysis of soil samples by the unweighted pairwise grouping method of mathematical averages (UPGMA, Dice co-efficient of similarity). (a) PCR-DGGE of DNA isolated by the Mo-Bio Ultra Clean Soil DNA Isolation Kit and (b) ) PCR-DGGE of DNA isolated by the Modified version of Duarte et al.(1998) (Bead Beat method). Sample numbers: Soil samples (2-13) originate from the landfill leaching experiment detailed in Chapter 4; with soil samples (14-16) the control samples that were leached with water only; soil sample (17) the original untreated soil; and samples (1 and 18) the DGGE marker and Bacillus soil isolate respectively.
(a) (b)