TABLE 1.
Colony mutation frequencies for various Mg2+ and dNTP concentrations in PCR-based lacZα complementation assay
| Expt no.a | Background CMFb,c | Mutation data under indicated conditions |
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|---|---|---|---|---|---|---|---|---|
| HIV RT |
MuLV RT |
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| 6 mM MgCl2 (5.6 mM free), 100 μM dNTPsc | 2 mM MgCl2 (1.98 mM free), 5 μM dNTPsc | 2 mM MgCl2 (1.6 mM free), 100 μM dNTPsc | 0.25 mM MgCl2 (0.24 mM free), 5 μM dNTPsc | 0.25 mM MgCl2 (0.07 mM free), 100 μM dNTPsc | 6 mM MgCl2 (5.6 mM free), 100 μM dNTPsc,d | 0.25 mM MgCl2 (0.24 mM free), 5 μM dNTPsc,d | ||
| 1 | 6/4,150, 1.4 | 32/3,637, 8.8 (7.4) | ||||||
| 2 | 6/4,600, 1.3 | 37/4,281, 6.9 (5.6) | ||||||
| 3 | 5/1,967, 2.5 | 27/1,961, 14 (12) | ||||||
| 4 | 13/4,560, 2.9 | 51/4,583, 11 (8.1) | ||||||
| 5 | 4/5,714, 0.7 | 30/10,362, 2.9 (2.2) | ||||||
| 6 | 24/8,276, 2.9 | 12/2,597, 4.6 (1.7) | ||||||
| 7 | 2/1,153, 1.7 | 16/2,137, 7.4 (5.7) | 6/2,164, 2.7 (1.0) | |||||
| 8 | 4/4,565, 0.8 | 38/8,050, 4.7 (3.9) | 30/10,362, 2.8 (2.0) | |||||
| 9 | 5/1,605, 3.0 | 10/1,041, 9.6 (6.6) | 13/1,354, 9.6 (6.6) | |||||
| 10 | 7/2,942, 2.4 | 26/3,318, 7.8 (5.4) | 13/3,585, 3.6 (1.2) | |||||
| 11 | 6/2,036, 2.9 | 10/1,358, 6.4 (3.5) | 5/1,310, 3.8 (0.9) | |||||
| 12 | 13/3,691, 3.5 | 20/2,224, 8.9 (5.5) | 6/1,503, 4.0 (0.5) | |||||
| 13 | 6/2,608, 2.3 | 16/1,639, 9.8 (7.5) | ||||||
| 14 | 6/2,998, 2.0 | 5/1,323, 3.8 (1.8) | 8/1,622, 4.9 (2.9) | |||||
| 15 | 7/2,464, 2.8 | 23/3,720, 6.2 (3.4) | 20/3,402, 5.9 (3.1) | |||||
| 16 | 4/2,611, 1.5 | 7/1,800, 3.9 (2.4) | 9/2,255, 4.0 (2.5) | |||||
| Avg ± SDe | 2.1 ± 0.8 | 10 ± 3.1 (8.3 ± 2.7) | 7.2 ± 2.4 (5.4 ± 1.3) | 8.5 ± 1 (5.7 ± 1.5) | 3.3 ± 0.9 (1.7 ± 0.5) | 3.8 ± 0.2 (0.9 ± 0.4) | 4.9 ± 0.9 (2.5 ± 0.8) | 4.9 ± 1.0 (2.8 ± 0.3) |
| P valuef | 0.157 | 0.110 | 0.003 | 0.006 | 0.017 | 0.019 | ||
| Relative fidelityg | 1 | 1.5 | 1.5 | 4.9 | 9.2h | 3.3 | 3.0 | |
Independent experiments performed at different times. In typical experiments, 1,500 to 3,500 colonies were scored for each condition.
In background assays, plasmid pBSM13ΔPvuII (Fig. 1B) was used as a template in PCRs to generate the insert that was scored in the assays. See Results for a discussion of the background calculation. Numbers shown are the colony mutation frequency (CMF, ×10−3), defined as white plus faint blue colonies divided by total colonies. Refer to Results and Materials and Methods for details.
The number of white plus faint blue colonies/total number of colonies is shown, followed by the colony mutation frequency (×10−3) for experiments under the listed conditions; this frequency minus the background frequency from column two is in parentheses.
Assays were performed with MuLV RT from New England Biolabs. All other reactions were with HIV RT.
Averages ± standard deviations from the experiments in the column are shown. Averages ± standard deviations of the CMF value minus the background frequency from the listed experiments are in parentheses.
Values were calculated using a standard Student t test and the background subtracted values from each condition. All values were scored against that at 6 mM Mg2+/100 μM dNTP.
All values are relative to the 6 mM Mg2+/100 μM dNTP average background CMF (8.3 × 10−3). The 0.0083 value was divided by the average CMF minus the background value for each condition to determine relative fidelity. Higher numbers indicate greater fidelity.
CMF values for this condition were on average less than 2 times the background. Hence, the values are near the limits of this assay with respect to quantitative reliability.