Table 1. Relative abundances of extension products by match or mismatch at the 3′-termini of primers.
Template |
|
Primer A |
Primer C |
Primer T |
Primer G |
P53-A | Modified primer | 0.7 ± 0.3 | 0.0 ± 0.2 | 100.0 ± 0.0 | 0.0 ± 0.1 |
Unmodified primer | 0.0 ± 0.1 | 43.5 ± 0.2 | 100.0 ± 0.0 | 7.9 ± 0.5 | |
P53-C | Modified primer | 0.2 ± 0.1 | 0.4 ± 0.2 | 3.5 ± 0.3 | 100.0 ± 0.0 |
Unmodified primer | 28.5 ± 0.4 | 1.0 ± 0.5 | 28.0 ± 0.2 | 100.0 ± 0.0 | |
P53-T | Modified primer | 100.0 ± 0.0 | 0.2 ± 0.1 | 1.4 ± 0.2 | 0.0 ± 0.3 |
Unmodified primer | 100.0 ± 0.0 | 47.2 ± 0.3 | 13.9 ± 0.4 | 54.8 ± 0.3 | |
P53-G | Modified primer | 0.0 ± 0.2 | 100.0 ± 0.0 | 4.4 ± 0.3 | 2.8 ± 0.3 |
Unmodified primer | 0.3 ± 0.2 | 100.0 ± 0.0 | 41.3 ± 0.5 | 2.5 ± 0.1 |
The switching by modified primers was almost perfect while undesirable strand extension reactions occurred with unmodified primers. The test samples were synthetic human P53 fragments each containing a different base, A, C, T or G, at the polymorphic site, with the four variants named P53-A, P53-C, P53-T and P53-G (n = 2), respectively. Modified means that a mismatched base was artificially introduced into the primer at the third position from the 3′-terminus; unmodified means that the primer did not include such a base. The data was normalized to the maximum intensity in a set of four experiments (primers A, C, T and G) as 100.