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
5.2.1.2 Quantification by spectrophotometry and PCR
Pair-wise t-tests showed that there were significant differences in DNA purity, yield and humic content between the two DNA isolation methods and the consequent Wizard™
Clean-Up (Promega) treatment of the Bead Beat isolated DNA (Table 5.1). The DNA yield attained attained using the Kit was significantly lower (P < 0.001) (average yield = 36.23 ng.µl-1) than that achieved with the Bead Beat protocol (average yield = 349.70 ng.µl-1).
Conversely, the purity achieved with the Kit isolation (average A260/A280 = 1.63) was significantly superior (P = 0.0026) to the Bead Beat protocol (average A260/A280 = 1.23).
A260/280 ratios close to 1.8 indicates DNA of higher purity whereas lower ratios are indicative of protein contamination. A260/A230 measurements indicated that humic contamination was greater for the Bead Beat protocol (0.29) than the Kit (0.63) (Figure 5.1), with the pair-wise t-test confirming a significant difference (P = 0.0007) in humic contamination between both treatments. Low A260/A230 ratios are indicative of humic acid contamination (Yeates, Gillings, Davison, Altavilla and Veal, 1998). Cullen and Hirsch (1998) reported that a heat treatment step (70 ºC – as in the case of this study) used in conjunction with bead beat protocols resulted in an increase in brown humic residue and lowered DNA yield. Similar findings were true for this study, with the exception that there was no increase in yield for the protocol devoid of heat treatment (Kit).
In this investigation, Bead Beat DNA extracts ranged from dark brown to beige in colour suggesting the presence of humic and fulvic acids, in addition to an organic soluble PCR inhibitor which is known to adversely affect DNA polymerase during PCR (Španová, Rittich, Štyriak, Štyriaková and Horák, 2006). The presence of large amounts of humic acid in the isolated DNA can also give rise to errors during the calculation of DNA yield (Steffan, Goksøyr, Bej, and Atlas, 1988). DNA yield estimations by A260 measurement can account for as much as a 10 fold overestimation in the presence of elevated humic acid contamination (Cullen and Hirch, 1998; Lloyd-Jones and Hunter, 2001). Bearing this in mind, treatment of the bead-beat isolated DNA with the Wizard™ Clean-Up columns was found to significantly increase (P = 0.0048) the purity of the isolated DNA such that there was no longer a significant difference (P = 0.1736) in DNA purity as compared to the DNA isolated by the Kit.
However, there was a significant decrease (P = < 0.0001), as much as 25 times, in DNA yield after treatment with the Wizard™ columns (Figure 5.1).
Table 5.1 Pair-wise t-test comparison of purity (A260/A280), yield, and humic content (A260/A230) of DNA isolated using two different isolation methods and a DNA clean-up step.
DNA Purity Factors Compared
P-value Significance Rating
Bead Beat vs Kit 0.0026 ***
Bead Beat vs Wizard 0.0048 ***
Kit vs Wizard 0.1736 ns
DNA Yield
Bead Beat vs Kit <0.0001 ***
Bead Beat vs Wizard <0.0001 ***
Kit vs Wizard 0.0496 *
Humic Content
Bead Beat vs Kit 0.0007 ***
Bead Beat vs Wizard 0.0018 ***
Kit vs Wizard 0.0251 *
***significant at P = 0.01; ns not significant (based on duplicate extractions) Bead Beat = modified method of Duarte et al. (1998)
Kit = MoBio Ultra Clean™ DNA Isolation Kit
Wizard = modified method of Duarte et al. (1998) plus the use of Wizard™ Clean-up columns
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
Kit Extraction Bead Beat Ex. Wizard
Method of DNA Extraction
A260/A280, A260/A230, DNA yield (ng/ul * 100)
A260/A280 DNA Yield A260/A230
b a
c
a
c b a
b a
Figure 5.1 Purity (A260/A280 and A260/A230) and yield (A260) measurements of DNA isolated from soil arrays using two DNA isolation methods [MoBio Ultra Clean™, Modified Bead Beat version of Duarte et al. (1998)], and Wizard™ Clean-up columns as a clean-up step.
The difference in yield was also less significant (P = 0.0496) between the Wizard™
treated DNA and the Kit isolated DNA. Robe et al. (2003) also noted that DNA yields from soil isolations were significantly reduced after purification and they found that this had a negative impact on the amplification of DNA sequences that were present in low numbers.
The purity and yield of the isolated DNA are fundamental to achieving success with subsequent downstream molecular manipulations, in this case PCR and DGGE (Picard et al., 1992; Cullen and Hirsch, 1998; Krsek and Wellington, 1999). DNA purity has a major bearing on the successful amplification of the DNA template during PCR (Cullen and Hirsch, 1998; Krsek and Wellington, 1999). Attempts to amplify the DNA isolated by the bead beat protocol proved unsatisfactory even after successive dilutions (1:10 to 1:1000) of the isolated DNA (result not shown). Negligible product was achieved for only a few samples at these dilutions and these were found to be unsuitable to generate DGGE profiles. Similar amplification constraints have been reported by Erb and Wagnerdöbler (1993).
High levels of humic material present in isolated DNA extracts have been shown to contribute significantly to the failure of subsequent PCR amplifications (Cullen and Hirsch,
1998; Lloyd-Jones and Hunter, 2001; He, Xu and Hughes, 2005). Moreover, the presence of phenolic compounds and heavy metals (which are common constituents of landfill leachate) in soil contribute to the inhibition and / or decrease in the sensitivity of subsequent PCR reactions (Robe, Nalin, Capellano, Vogel and Simonet, 2003; de Lipthay, Ezinger, Johnsen, Aamand and Sørensen, 2004). Protein impurities in soil-DNA extracts also have a negative impact on PCR amplification (Krsek and Wellington, 1999).
Purification of the Bead Beat DNA extract with Wizard™ Clean-up columns was considered an essential pre-treatment to render DNA purity suitable for satisfactory PCR amplification. This approach is well documented, although the exact commercial clean-up columns may differ (Duarte et al., 1998; Niemi et.al., 2001). Wizard™ column pretreated DNA was successfully amplified indicating that a clean-up step was essential.
A PCR product of approximately 200 bp (theoretically 180 bp) was clearly visible for reactions incorporating DNA from both isolation methods as template (Plate 5.2). The kit isolated DNA produced clearly defined products with no evidence of unused primer (Plate 5.2a). After PCR amplification using Bead Beat DNA as template for the reaction, the resulting product contained a larger amplified artefact (approximately 300 bp) in addition to the target segment (Plate 5.2b). However the product generated by the target sequence was visibly more substantial than this artefact. Furthermore there was a larger amount of unused primer.
Plate 5.2 Amplification of 16S rDNA fragments from soil samples of array Ah demonstrating the effect of DNA method on the quality of PCR product. (a) Mo-Bio Ultra Clean DNA isolation kit, lanes: 100 bp DNA ladder (Promega) (1 and 14); samples 1 to 12 (samples of array Ah) (2 – 13); wet controls (C1; C2; C3) (15 - 17); dry control (Cd) (18); DGGE marker (19); positive control (Bacillus isolate) (20); and negative control (21) . (b) Bead beat isolation, lanes correspond to those described in (a).
1 500 500 200
1 2 3 4 5 6 7 8 9 10 11 12 13 1 2 3 4 5 6 7 8 9 10 11 12 13
14 15 16 17 18 19 20 21 14 15 16 17 18 19 20 21
(a) (b)
bp
artefact
unused primer
5.2.1.3 Influence of DNA extraction method on bacterial community structure and