Las soluciones vienen por el conocimiento

5.8.1 WGA using the REPLI-G® kit

Whole genome amplification has been shown to be effective in amplifying gDNA isolated from individual cells (Raghunathan et al., 2005). Consequently, large amounts of high molecular weight DNA structures, referred to as wgaDNA, are generated through the random amplification events using random primers and high fidelity polymerase such as Φ29 DNA

Culture-dependent screening of novel nanoarchaeal species

154 polymerase (Aviel-Ronen et al., 2006). Due to minute amounts of gDNA were extracted from the enriched cell cultures, this approach was adopted to amplify any DNA extracted with minimal amplification bias. The wgaDNA could then be used for large insert cloning.

WGA was conducted on gDNA from enriched samples using the REPLI-G® kit (QIAGEN) according to the manufacturer‘s instructions. The gDNA was successfully amplified, resulting in high molecular weight DNA (Figure 5.8). TAT2-2, SHS-G, positive control and the negative control samples yielded 69, 78, 75 and 72 µg, respectively (Figure 5.8).

Figure 5.8: WGA using the REPLI-G® kit. Lanes 1 and 11: 100 ng fosmid control DNA; lane 3: TAT2-2; lane 5: SHS-G (control); lane 7: control DNA from the kit; lane 9: negative control; lanes 2, 4, 6, 8 and 10 were left empty to allow space for gel slicing.

Owing to the sensitivity of this method, even trace amounts of contaminating DNA can be amplified, and the formation of primer dimers can result in background synthesis. As shown in Figure 5.8, high molecular weight DNA was obtained. However, the negative control (lane 9) produced equally large yields of high molecular weight DNA. The generation of a high DNA background in the negative control has been observed in previous studies, and is believed to be caused by artifactual synthesis such as self-priming or primer dimer formation, DNA

155 contamination or both (Raghunathan et al., 2005; Hutchison & Venter, 2006; Zhang et al., 2006).

5.8.2 WGA using Φ29 DNA polymerase

To overcome the background formation caused by the REPLI-G® kit, Φ29 DNA polymerase was used with modified random heptamers as primers to avoid primer re-annealing, and was found to be effective in both the synthesis of high molecular weight DNA and in removing the background amplification (Figure 5.9). However, the amplification yield using modified random heptamers as primers was quantitatively less compared to REPLI-G amplification, and can be attributed to the absence of primer dimer formation, and the quality and quantity of the DNA template used.

Figure 5.9: Amplification of gDNA from enriched culture samples using Φ29 DNA

polymerase. Lane M: λ PstI DNA marker; lane 1: TAT9-1; lane 2: TAT10-1; lane 3: negative control.

Even though no quantifiable amounts of DNA were observed from the eluate of cell fractions separated by flow cytometry, the eluate was also used as template for MDA, so that any trace

Culture-dependent screening of novel nanoarchaeal species

156 amounts of DNA present could be amplified. However, only enriched culture samples could amplify with Φ29 DNA polymerase, suggesting that high quality DNA template was most efficient for successful MDA. Previous reports have indicated that the Φ29 DNA polymerase can efficiently amplify from as little as 0.03 ng DNA template (Dean et al., 2002). On the basis of the results obtained in this study, however, this has not been the case. The template DNA is most like to have been the main limiting factor contributing to the failure of WGA, including other factors such as DNA polymerase inhibitors.

5.8.3 Purification of wgaDNA

A significant amount of wgaDNA was lost during purification using low-melting-point agarose. In the preparation of fosmid libraries, at least 2.5 μg of 40 kb DNA fragments are required for efficient library construction. In various amplification reactions, 1-6.9 μg was obtained. Gel electrophoresis showed that wgaDNA products migrated below the 40 kb fosmid control DNA benchmark; they were about 11.5 kb and not suitable for fosmid library construction (Figure 5.9). In order to avoid low cloning efficiency and the formation of chimeric clones, wgaDNA products could not be used for fosmid library construction.

Tools capable of generating data from single cells are limited because of the difficulties involved when dealing with small cell quantities and volumes to be analysed, including the disruption or lysis of cells (Brown & Audet, 2008). Cell lysis at a small scale or at a single-cell level can be a challenge especially when high cell dilutions are used, due to the possibility of the loss of cells during lysis. To circumvent this problem, special devices and protocols have

157 been developed for the control and handling of picolitre cell volumes (Irimia et al., 2004). Unfortunately, it was not possible to implement these methods in this study.

5.8.4 16S rRNA gene amplification from wgaDNA

In order to test whether the wgaDNA was efficient for other molecular applications such as PCR, the wgaDNA was amplified with nanoarchaeal targeting primers, A571Fb/N989R. No nanoarchaeal amplicons were detected from wgaDNA derived from enriched culture samples. However, amplification was obtained with a control wgaDNA of environmental sample (wgaDNA extracted from environmental samples) SHS-G from the Ethiopian Shalla hot springs (Figure 5.10), proving that wgaDNA can be used successfully for other PCR-based applications and to recover sequences of taxonomic groups of interest.

Figure 5.10: 16S rRNA gene amplification using wgaDNA as a template. Lane M: DNA

marker; lane 1: SHS-G; lane 2: nanoarchaeal clone T173 as positive control; lane 3: negative control.

Culture-dependent screening of novel nanoarchaeal species

158 Detection of a nanoarchaeal PCR signal from environmental wgaDNA, but not from enriched samples, suggests that the nanoarchaeal cells might have been lost during cultivation and/or DNA extraction. Alternatively, nanoarchaeal cells might have been present in such low amounts that they could not be detected by amplification.