2. PROSPECTING FOR THE ACID PHOSPHATASE GENE FROM THE MYCO-
2.4 DISCUSSION
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72 was one of several factors that made designing of PCR primers using these sites/sequences difficult. These factors include low homology, the presence of unfavourable amino acids such as serine within the conserved sequences, the absence of continuous conserved amino acids and the shortness of the conserved sites, 6 AA or less (Figure 2.3).
2.4.2 DNA extraction and PCR optimization
The data indicate that optimum DNA yield can be obtained from Cladonia portentosa using commercial extractions kits as well as conventional methods (Figures 2.4-2.5). In this study, the quantity of DNA isolated by commercial kits were generally lower compared to the traditional methods, although higher yield have been reported by GRIFFITHS et al. (2006) using a commercial kit. Low DNA yield obtained in using commercial kits have been attributed to the fact that some DNA molecules failed to bind to silica particles and were lost during washing or could not be eluted because of irreversible bonds (BOOM et al., 1990). Traditional DNA protocols used in this study were labour-intensive. However, data indicated that these two methods were highly efficient in extracting high yields of genomic DNA in Cladonia portentosa and other fungi used in this study. Higher DNA yield increases the chances of detecting rare species (WINTER et al., 1980).
Since the aim of the PCR was to clone the gene that was virtually unknown, low temperature stringencies were initially used in the PCR (42º to 52 ºC). The annealing temperature 42 ºC was low enough to guarantee efficient annealing of the primer to the target, but high enough to minimize non-specific binding (Figure 2.7). Later, the annealing temperature was increased to 54-60 ºC. Distinct bands were obtained from the positive controls but none were obtained from C. portentosa (Figure 2.8). Failure to amplify the apase gene by PCR using C. portentosa genomic DNA was intriguing and perplexing. Several attempts to optimise varying PCR parameters were conducted.
Visual assessment of DNA quality showed it was sufficient for PCR irrespective of the extraction method (Figures 2.4-2.5). Expected PCR products were amplified in both positive controls (A. niger and N. fischeri) using the reported extraction protocols. Most conventional
73 methods use toxic and hazardous reagents such as phenol and chloroform to separate cellular debris from the DNA, thus, the possible contamination of isolated nucleic acid by these toxic chemical cannot be ruled out (GRIFFITHS et al., 2006; NIU et al., 2008). In addition, the use of high salt concentration buffers and proteinase K treatments of DEAE-cellulose to obtain a higher purity of DNA in several non-toxic extraction methods have been linked to compromised PCR activities (ALJANABI and MARTINEZ, 1997; DE LA CRUZ et al., 1997; SHARMA et al., 2000; BULDEWO and JAUFEERARLLY-FAKIM, 2002; ANGELES et al., 2005).
In addition, the lack of PCR product has been linked with contaminants such as tannins, polysaccharides and pigments that can inhibit the annealing of DNA or the enzymatic activity of restriction endonucleases (PANDEY et al., 1996; ROGSTAD, 2003).
PCR amplification was further conducted using varying concentrations of genomic DNA extracted by three techniques (to eliminate all the possible inhibitors of lichen DNA). Studies by WANG et al. (1989) showed that high template concentration occurs as a result of the PCR amplifications, phenomena known “as a substrate saturation of enzyme”, or product inhibition of enzyme, incomplete strand separation and product strand re-annealing can be limiting factors for efficient amplification (WANG et al., 1989). No PCR products were obtained using genomic DNA of Cladonia portentosa irrespective of the DNA extraction method. The successful amplification of PCR products from positive control organisms revealed that the DNA isolated using three methods were suitable for PCR application.
Other variables that could influence the efficiency of the PCR amplification were studied:
These parameters included the concentrations of dNTPS, MgCl2, primers, Taq polymerase and the PCR cycle profile (WANG et al., 1989). A difference in primer efficiency is a difficult parameter to regulate for quantitative analyses (WANG et al., 1989). The degenerate primers were designed using sequences of close relatives of C. portentosa, mainly from the Ascomycete family. To improve specificity and minimize the degeneration, codon usage tables were consulted for base preference in different organisms (INNIS and GELFAND, 1990). Inosine was used which pairs with all bases. In terms of primer concentration, 40 and 80 pmol were sufficient to amplify PCR products in both control organisms (Figures 2.7 and 2.8).
Recommended primer concentration ranging from 1.0 µM to 3.0 µM were used since many of
74 the primers in the degenerate mixture are not specific to the target (SHYAMALA and AMES, 1993). In either case, mismatches in oligonucleotide annealing are typically limiting (RUBIN and LEVY, 1996).
Different polymerase enzymes were tried (Taq polymerase, Faststart Taq polymerase and Phusion Taq polymerase), since several studies show that the choice of enzyme can affect yield (INNIS and GELFAND, 1990). FastStart Taq polymerase became the enzyme of choice in this study. The highest length of the expected fragment in this study was limited to 1500 bp. It has been shown that the amount of amplified DNA fragment in a given sample has a prevailing influence on the amplification efficiency (LARZUL et al., 1990).
Magnesium concentration was also optimized. The concentration (MgCl2) of 2.5 µM was optimal but no success was obtained for C. portentosa (Figure 2.9). Magnesium affects several aspect of PCR including DNA polymerase activity, which can affect yield, primer annealing, and specificity (INNIS and GELFAND, 1990). Higher concentrations of free magnesium can result in greater yield, but can also increase non-specific amplification and reduce fidelity (ECKERT and KUNKEL 1990; WILLIAMS, 1989). Since PCR products were amplify from positive control organisms, it was concluded that failure to amplify the apase gene from C.
portentosa was not the result of the reagents or genomic DNA.
In order to avoid time-consuming trial-and-error testing using degenerate primers, attempts to purify the apase were made. It was anticipated that the partial sequence of the purified enzymes would provide a corresponding apase gene (KEROVUO et al., 1998; CHO et al., 2005).
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