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
5.2.1.4 Assessment of bacterial community diversity
Beat method. Overall, sample for sample, the PCR-DGGE amplicons of DNA isolated by the Kit showed greater diversity than that generated by the Bead Beat method (Figure 5.3a). This superior diversity was confirmed by lower D indices recorded for the kit isolated DNA amplicons (Figure 5.3b) which effectively reveals that there is a lower probability that two species (bands) identified in a community (sample) will be the same species, thereby reflecting a higher diversity (Krebs, 1985; Edwards et.al., 2001). The difference identified between the means of H’ for both treatments were confirmed by significantly different mean D values (P = <0.001). This study made use of two diversity measures (Shannon-Weaver Diversity Index and Simpson’s Index) and their related equitability equations so as to increase the validity of the conclusion drawn from this study (Kocherginskaya et al., 2001).
Each diversity index distributes a different weighting in each equation to composite species. The Shannon-Weaver Index gives more weight to bands with relatively low signal intensities while the Simpson’s Index tends to apply additional weight to dominant bands with relatively high signal intensities (Hill et al., 2003). Essentially, Simpson’s index measures the diversity of the most numerically predominant amplicons which are the direct products of the selected DNA isolation methods (Kocherginskaya et al., 2001). Thus, by calculating these indices both possible extremes, which may be encountered in any sample, can be taken into account.
The lower evenness indices measured for the PCR-DGGE amplicons of DNA isolated by the Bead Beat method indicate the presence of a greater number of numerically dominant species (bands) isolated by this method (Figures 5.4a and b). This method favoured the extraction of bacterial DNA from certain species over others. This was supported by average EH and ED values of 0.92 and 0.69 respectively. Conversely, the Kit method showed evenness indices closer to one (average EH=0.95and ED=0.78), indicative of the isolation of a more equitable or even distribution of all extracted bacterial DNA per soil sample. Consequently, significant differences were recorded between the mean EH (P = 0.002)and ED (P = 0.002) of both treatments. The EH and ED ranged from 0.97 to 0.93 and 0.85 to 0.67 respectively for the DNA isolated by the Kit method and 0.96 to 0.87 and 0.80 to 0.49 respectively for the Bead Beat method. The broader range recorded for both evenness indices in relation to the Bead
Beat method implies a greater variability experienced in reproducing the isolation of similar proportions of DNA across the samples. An evenness index of one indicates a proportionate distribution of microorganisms (Camargo et al., 2005).
(a)
0 0.5 1 1.5 2 2.5 3 3.5
0 10 20 30 40 50
Approximate depth of samples in microcosms (cm)
Shannon-Weaver Index (H')
(b)
0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16
0 10 20 30 40 50
Aproximate depth of samples in microcosms (cm)
Simpson's Index (D)
Figure 5.3 Diversity analyses of data generated by PCR-DGGE of DNA isolated using a Mo-Bio Ultra Clean Soil DNA Isolation Kit (Kit method) (▲) and DNA isolated using a Modified version of Duarte et al.(1998) (Bead Beat method) (□). Soil samples were taken from soil array Ah perfused with synthetic landfill leachate over 12 weeks (Chapter Four). The generated DGGE data was applied to (a) Shannon-Weaver Diversity Index and (b) Simpson’s Diversity Index.
(a)
0.86 0.88 0.9 0.92 0.94 0.96 0.98
0 5 10 15 20 25 30 35 40 45
Aproximate depth of samples in microcosms (cm) Shannon-Weaver Evenness Index (EH)
(b)
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
0 5 10 15 20 25 30 35 40 45
Aproximate depth of samples in microcosms (cm) Simpson's Equitability Index (ED)
Figure 5.4 Diversity analyses of data generated by PCR-DGGE of DNA isolated using a Mo-Bio Ultra Clean Soil DNA Isolation Kit (Kit method) (▲) and DNA isolated using a Modified version of Duarte et al.(1998) (Bead Beat method) (□). Soil samples were taken from soil array Ah perfused with synthetic landfill leachate over 12 weeks (Chapter Four). The generated DGGE data was applied to (a) Shannon-Weaver Evenness Index and (b) Simpson’s Evenness Index.
Commercial DNA isolation kits, exclusively designed for the isolation of microbial DNA from soil and sediments, offer a convenient method that is quick, simple, routinely reproducible, and appropriate for successive DNA isolation reactions. However, caution must be exercised when relying exclusively on single isolation methods since there are bound to be recalcitrant bacteria present from which DNA is not readily isolated. Any given protocol will favour the isolation of DNA from bacteria most susceptible to the physico-chemical methods employed thereby contributing significantly to the composition and diversity of the prevalent microbial community (Roose-Almsaleg, Garnier-Sillam, and Harry 2001). The choice of DNA isolation method revolves around numerous factors; these include the efficiency of isolation and purification, the quality of the isolated DNA for downstream reactions, and the representative constitution of the isolated DNA in any given sample. Roose-Almsaleg et al.
(2001) stated that the choice of a DNA isolation protocol centres on a compromise between quality, representative composition, and destined applications of the isolated DNA while taking into account that each isolation technique suffers some form of bias or limitation. It is imperative that each isolation technique is adapted for the type of sample in question (Zhou, Bruns and Tiedje, 1996). Since it is not only the parent sample that will influence the extraction efficiency of the technique but also the subsequent treatments that the sample is exposed to will have a significant bearing on the efficiency of the DNA isolation technique.
Dubey, Tripathy, and Upadhyay (2005) stated that an ideal DNA isolation technique would;
process all samples uniformly, isolate DNA from all the members of a soil microbial community, process multiple samples over a satisfactory time scale, and produce DNA of satisfactory yield and purity. Additionally, the quantity of DNA isolated will determine, in some measure, the numerical diversity and numerical dominance of autochthonous bacteria in the soil sample but not necessarily the functional dominance in the community (Krsek and Wellington, 1999).