TABLE 6.

Mismatched primer extension at 0.25 or 2 mM Mg²⁺

Base pair at the 3′ enda	[Mg2+] (mM)b	Vmax (%/min)c	Km (μM)d	Vmax/Kme	Standard extension efficiencyf	Relative fidelityg	P valueh
C·G	2	21.4 ± 2.0	0.37 ± 0.13	57.8 ± 19.2	1
	0.25	24.1 ± 10.8	0.41 ± 0.01	58.8 ± 28.1	1
G·C	2	39.9 ± 14.5	0.27 ± 0.13	147.8 ± 40.6	1
	0.25	18.7 ± 3.7	0.14 ± 0.03	133.6 ± 14.3	1
C·T	2	21.2 ± 2.7	500 ± 181	(4.2 ± 1.8) × 10−2	(7.3 ± 3.3) × 10−4	1	0.013
	0.25	3.1 ± 0.42	748 ± 57	(4.1 ± 0. 59) × 10−3	(7.1 ± 1.0) × 10−5	10
C·A	2	11.1 ± 2.9	445 ± 188	(2.5 ± 0. 57) × 10−2	(4.3 ± 0.94) × 10−4	1	5.2 × 10−5
	0.25	4.4 ± 3.9	1,546 ± 714	(2.8 ± 1.9) × 10−3	(4.8 ± 3.4) × 10−5	8.9
C·C	2	0.70 ± 0.29	77 ± 14	(9.1 ± 2.2) × 10−3	(1.6 ± 0.38) × 10−4	1	0.027
	0.25	0.20 ± 0.01	61 ± 34	(3.3 ± 2.1) × 10−3	(5.6 ± 3.6) × 10−5	2.9
G·T	2	18.6 ± 3.3	157 ± 44	0.12 ± 0.03	(8.1 ± 2.5) ×10−4	1	0.13
	0.25	8.6 ± 1.5	120 ± 6	(7.2 ± 1.4) × 10−2	(5.4 ± 1.1) × 10−4	1.5
G·A	2	0.95 ± 0.04	196 ± 50	(4.8 ± 1.6) × 10−3	(3.2 ± 1.0) × 10−5	1	0.23
	0.25	0.50 ± 0.30	169 ± 13	(2.9 ± 1.6) × 10−3	(2.2 ± 1.3) × 10−5	1.5

^aRefer to the mismatch extension constructs in Fig. 3. In this assay, primers with a matched C·G or a mismatched C·T, C·A, or C·C at the 3′ end were extended on one construct. A second construct with a matched G·C or mismatched G·T or G·A was also used.

^bThe concn of free Mg²⁺ in the reactions is shown. Free Mg²⁺ was calculated as described in Materials and Methods using the dissociation constant for Mg²⁺ and ATP.

^cV_max is the maximum velocity of extending each primer-template construct. See Materials and Methods for a description. Values are averages from at least 3 experiments ± standard deviations.

^dK_m of the next correct nucleotide being added (dCTP for C·G, C·T, C·A, and C·C or dGTP for G·C, G·T, and G·A extensions). Values are averages from at least 3 experiments ± standard deviations.

^eValues are averages from at least 3 experiments ± standard deviations.

^f[V_max/K_m (mismatch)]/[V_max/K_m (match)] at each respective Mg²⁺ concentration. Values are averages from at least 3 experiments ± standard deviations.

^gValues are relative to that at 2 mM. Determinations were made by dividing the standard extension efficiency at 2 mM Mg²⁺ by the extension efficiency at 0.25 mM Mg²⁺. Higher values indicate greater fidelity.

^hValues were calculated using a standard Student's t test. Misinsertion ratio values from experiments at 0.25 mM Mg²⁺ were compared against that under the 2 mM Mg²⁺ condition listed above for each mismatch type.