3. Results and Discussion
3.3. Capillary Gel Electrophoresis
3.3. Capillary Gel Electrophoresis
As working with radioactivity has obvious disadvantages, I switched to fluorescently labelled primers for single-nucleotide incorporation primer extension experiments. Despite its utility, analysis of those experiments by denaturing PAGE has several disadvantages (see 1.5. Capillary Electrophoresis).
Those drawbacks can be overcome if the DNA polymerase catalysed extension of fluorescently labelled primers is analysed using CE. Thereby, the throughput can be dramatically increased, as CE offers the possibility for multiplexing. Since this method is by default used for Sanger DNA sequencing, microsatellite analysis or single nucleotide polymorphism analysis,[62, 115] it was necessary to adopt this method to meet my requirements.[137]
First, separation of short, fluorescently labelled DNA oligonucleotides was optimised. Therefore, two FAM labelled oligomers (21mer and 22mer) were purchased. Conditions applied during separation were modified and adapted until single base resolution of the oligomer mixtures was ensured (Figure 22). Run parameters established for this separation are listed in 6.2.9. Capillary Electrophoresis
Capillary Electrophoresis separates fluorescently labelled oligonucleotides by size and charge as they migrate through a polymer filled capillary. Single nucleotide incorporation primer extension experiments are conducted as described (6.2.6. Primer Extension Assay[136]). Instead of radioactively labelled primers, FAM labelled ones are employed and reactions are stopped by addition of stop solution without bromophenol blue and xylene cyanol (80 % (v/v) formamide, 20 mM EDTA). CE samples are prepared by mixing 10 µl LIZ size standard diluted 1:80 in HiDi formamide (Applied Biosystems) and 10 µl fluorescently labelled reaction mixture (after addition of stop solution diluted 1:10 with HiDi formamide, to dilute EDTA). If multiple reactions are to be analysed in parallel, those are pooled in one well prior to injection. The reaction sample and size standard are injected electrokinetically into a 50 cm capillary array filled with Performance Optimised Polymer (POP6). High voltage electrophoresis (15 kV) over 180 s ensures single base resolution. Typical run parameters are depicted in Table 4.
Figure 22: Separation of fluorescent primers using CE. a) Migration behaviour of a FAM labelled 21mer (DNA) analysed by capillary electrophoresis;; b) migration behaviour of a FAM labelled 22mer (DNA) analysed by capillary electrophoresis;; c) migration comparison of different mixtures (2/1;; 1/1;; 1/2) of a 21mer and 22mer (DNA) to prove sufficient resolution for analysis of single - nucleotide incorporation primer extension reactions.
orange: size standard (LIZ 120), blue: FAM labeled primers.
The established assay was applied for single-nucleotide incorporation primer extension experiments. Therefore, processing of dGTP by the DNA polymerase 9°North exo- was examined by the usage of fluorescently labelled primers and analysis by CE (see Figure 23 a - c). Radioactively labelled primer extension experiments and separation by PAGE (see Figure 23d) was performed additionally to allow comparison between both methods.
Comparison of the quantitative evaluation of both analysis methods showed that the results are very similar. Therefore, evaluation of incorporation of the modified nucleotides by DNA polymerase 9°North exo- will be examined by CE. To take full advantage of the possibilities for automating supplied by employing CE, the described assay was adapted to allow the analysis of several reactions in one capillary and one run. In this approach single-nucleotide incorporation primer extension experiments are analysed. Thus, several reactions can be separated during one run in one capillary, if those extension reactions are performed using fluorescently labelled oligomers of different sizes. As depicted in Figure 23e sufficient separation could be ensured, if those primers were designed with a size difference of 8 nucleotides varying in size from 21 to 55 nucleotides.
With this experimental setup, it is possible to analyse 5 reactions in one run and one capillary in parallel enhancing the through put. The principles of this assay can be applied to further scale up the through put. Processed primers can be labelled using different dye-labels having well separated excitation and emission spectra (e.g. FAM, NED, VIC and PET). Additionally, longer primers can be used for analysis. Simultaneous analysis of multiple substrates or reaction conditions can therefore be enabled by multiplexing oligonucleotide design by size and fluorescent dye.
3. Results and Discussion 50
Figure 23: Capillary electrophoretic and PAGE analysis of single-nucleotide incorporation primer extension experiments of dGMP employing DNA polymerase 9°North exo-. a) Opposite a template containing C;; b) opposite a template containing C;; c) quantitative analysis of single-nucleotide primer experiments depicted in a (black) and b (grey);; d) PAGE analysis and quantitative evaluation of single-nucleotide incorporation primer extension experiments of dGMP employing DNA polymerase 9°North exo-;; e) capillary electrophoretic analysis of 5 different single-nucleotide incorporation experiments employing different lengths of FAM labelled primers in parallel. 50 µM dGTP and 10 nM 9°North exo- were used;; reactions were stopped after indicated time points. Experiments were done at least in triplicates.
3.3.1. Discrimination of 5mC by Emplyoing Modified Nucleotides and 9°North DNA Polymerase
Testing all in position 6 modified dGTP derivatives for incorporation opposite C and 5mC employing the second B family DNA polymerase 9°North, it can be observed that all nucleotides are successfully
processed (see Figure 24). As already observed before, incorporation efficiencies of the dGMP analogues decrease by increasing size of the introduced modification. Additionally, incorporation opposite C is favoured for all nucleotides over incorporation opposite 5mC. Again, discrimination can already be observed for processing of the unmodified dGTP. This discrimination ratio enhances by the usage of some of the modified nucleotides. In contrast to the results obtained by employing the DNA polymerase KOD exo-, the alkylation of dGTP in position 6 (1a - 1d) does not improve discrimination behaviour. Instead, the amino-modified nucleotides (9a - 9o) show remarkable improvement in differences in incorporation efficiencies using 9° North exo-. The highest discrimination rates can be observed by employing nucleotides 9b, 9c or 9n (see Figure 24 and Figure 25).
Figure 24: Structures of modified dG*TP analogues including % incorporation opposite C or 5mC employing DNA polymerase 9°North exo- in single-nucleotide primer extension experiments. 50µM dGTP or dG*TP and 10 nM 9°North exo- were used, reactions were stopped after 10 min. Experiments were done at least in triplicates.
Arithmetic mean is given;; errors are given in the appendix.
But still, more pronounced discrimination behaviour can be observed by employing the DNA polymerase KOD exo- in combination with nucleotide 1b. Even if the discrimination ratio is not further improved by usage of the second B family DNA polymerase 9°North exo-, we proved the tendencies observed in previous studies: this DNA polymerase was capable to incorporate all in position 6 modified nucleotides. Additionally, discrimination can already be observed by incorporation of unmodified dGMP and this discrimination can be further improved by processing of modified nucleotides.
3. Results and Discussion 52
Figure 25: Incorporation experiments of modified nucleotides leading to most pronounced differences in incorporation efficiencies employing DNA polymerase 9° North exo-. a) Partial primer / template sequence used.
b) PAGE analysis and quantitative evaluation of single-nucleotide incorporation primer extension experiments of dGMP and nucleotides 1b, 9b, 9c and 9n opposite a template containing C (black) in comparison to a template containing 5mC (grey) employing the DNA polymerase 9°North exo-. 50 µM dGTP or dG*TP and 10 nM 9°North exo- were used;; reactions were stopped after indicated time points. Experiments were done at least in triplicates.