Results and Discussion
Analysis of the Hae III restriction fragments of ᵩX174
The ᵩX174 is a small, single-stranded DNA virus that infects Escherichia coli. It is also called a bacteriophage. It was the first genome to be sequenced. 3 Its replicative form (RF) is a double stranded circular DNA molecule of 5386 base pairs (bp). When it is digested by a restriction enzyme Hae III, 11 DNA fragments are generated.4 These fragments have been used as popular DNA standards for size determination of DNA fragments in the past 40 years. It is available from SCIEX as dsDNA 1000 Test Mix (PN 477414). Figure 9a shows the restriction map of ᵩX174 with Hae III sites, the origin of replication, and location of 11 genes indicated. Figure 9b is a representative electropherogram obtained by separation of the Hae III fragments of ᵩX174 on PA800 Plus using dsDNA 1000 gel, LIFluor EnhanCE dye and coated DNA capillary at 40.2 cm total length. Instrument set up and separation conditions were described in the Methods section. All 11 fragments including two fragments differing by 10 bps were baseline resolved within a 25 minutes run. As shown in Figure 10, a calibration curve was generated with results from Figure 9b. The best curve fitting was obtained with the quartic (4th degree polynomial) model with excellent R square values: 0.9992 at the linear scale (panel A) and 0.9997 at log scale (panel B).
Further experiments were carried out to determine the limit of detection (LOD) and limit of quantitation (LOQ) as well as the linearity of detector response to concentration of the DNA sample in this method. The Hae III fragments of ᵩX174 were serially diluted by 2 fold each time and separated. Figure 11A shows the electropherograms obtained with some of the diluted samples. The concentration of the 872 bp fragment is indicated next to each electropherogram trace. The signal to noise ratio for the 872 bp peak was 5.4 at the concentration of 0.79 ng/ml, and 16.1 at 1.6 ng/ml. Therefore, the LOD is 0.79 ng/ml and the LOQ is around 1 ng/ml. Figure 11B shows that good linearity was obtained when plotting the corrected peak area of the 872 bp fragment against its concentration from 0.79 ng/ml to 404 ng/ml. The R square value was 0.995. The dynamic range was 2.7 log.
Analysis of DNA fragments over extended size range
Many of the vectors used in producing nucleic acid therapeutics for gene and cell therapy as well as for DNA vaccination have sizes well over 10 kb. Restriction enzyme digestion of these large vectors often generates some fragments that are larger than 1 kb as well as fragments that are smaller than 500 bps. Analysis of these fragments over an extended size range requires the use of DNA size standards that provide a long size range coverage. Several DNA standards with broader range of size coverage were tested. Among them, the 1 kb DNA ladder and 1 kb plus DNA ladder from Thermo were selected.
Analysis with 1 kb DNA ladder
The 1 kb DNA ladder includes 12 large fragments created by repeating a 1018 bp fragment for 1 to 12 times. It also contains additional 11 fragments with sizes from 75 bp to 1636 bp that are generated by Hinf I digestion of a plasmid vector pBR322. For the 1 kb DNA ladder, the best separation was obtained with the separation voltage at 6 kv and a 40 cm effective length DNA capillary. As shown in Figure 12, an experiment was done to test method repeatability. The 1 kb DNA ladder at the concentration of 20 ng/ul was injected at 0.5 psi for 10 sec for 8 runs and separated using dsDNA 1000 gel and LIFluor EnhanCE dye. Each separation was performed at a voltage of 6 kv for 65 minutes with 20 psi pressure at both capillary ends. Results in Figure 12 showed that the peak patterns were consistent between the 8 different runs. Fragments under 500 bp with size differences of 20 to 50 bp were well separated. The picture inset in Figure 12 shows a zoomed-in view of the area where large DNA fragments with sizes of 1.6 kb to 12 kb were baseline separated as well. Good repeatability was demonstrated by the RSD% values for the 1636 bp peak: 0.43% for migration time and 2.19% for corrected peak area.
Analysis with 1 kb plus DNA ladder
The 1 kb plus DNA ladder contains 18 fragments in the range of 100 bp to 15,000 bp. Two different methods were evaluated with this DNA ladder. The first one included using the DNA capillary at 30 cm effective length and electrokinetic sample injection. An experiment was done to simulate analysis of fragments produced by restriction enzyme digestion of a plasmid and a virus. The 1 kb plus DNA ladder (at 6.25 ng/µl), linearized plasmid pUC18dG (at 3 ng/µl) and Hae III fragments of ᵩX174 (at 2.5 ng/µl) were injected at 1 kv for 2 seconds. Separation was performed at 7.8 kv for 30 minutes. Results in Figure 2 demonstrated that excellent resolution was achieved with baseline resolution of fragments differing by 6-10 bp around 200-300 bp region, 30-50 bp around 800 bp region and 150 to 315 bp around 1 to 3 kb area. A calibration curve was created by plotting the size of DNA fragments in the 1 kb plus ladder against migration time using the quantitative analysis feature of the 32 Karat software. The R square value was 0.9982 at the log scale using the quartic model (data not shown). This calibration curve was used to deduce predicted sizes for the linearized pUC18dG and 10 of the Hae III fragments of ᵩX174. The predicted sizes were compared to their corresponding theoretical sizes (Table 1). The differences between predicted sizes and theoretical sizes were no more than 5 to 7% of the theoretical sizes for all fragments, demonstrating the accuracy of size determination for restriction fragments by this method. In addition, this method can be potentially useful for analysis of host cell DNA in cell culture-produced vaccines in which over 80% of residual DNA was under 1000 bp with the major peak around 150 bp and some minor peaks in the range of 1000 bp to 10 kb. 5-6
The second method with the 1 kb plus DNA ladder involves using the DNA capillary at 40 cm effective length and pressure injection for sample loading. Figure 13 shows results obtained by injecting a mixture of the 1 kb plus DNA ladder and the 1 kb DNA ladder at 0.5 psi for 10 seconds. Separation was carried out at 10 kv for 35 minutes. Large DNA fragments were separated with a resolution of 1 kb. In the region between 850 to 2000 bp, the resolution was 136 to 150 bp. Fragments smaller than 500 bp were well resolved when the size differences between them were 20 bp or larger. Interestingly, a 298 bp fragment was separated from a 300 bp fragment while a 396 bp fragment was not resolved from a 400 bp fragment. These differences may be due to differences in sequence composition or differences in interaction with the intercalating dye. Resolution achieved with this method was similar to that obtained with the first method.
Users can choose which method to use based on their samples.