The error rates for natural base pairing by proofreading-deficient DNA polymerases (without 3'-exonuclease activity) range from [10.sup.-3] to [10.sup.-6] error/bp.
Base pairing complementarity in polymerase reactions
Among these unnatural base pairs, the order of the efficiency and selectivity of the unnatural base pairing in T7 transcription is v-y > s-y > x-y.In comparison with the freely rotated 6-dimethylamino group of x, the planarity and the electronegative atoms of the 6-heterocycles, thiazolyl in v and thienyl in s,efficiently exclude the pairing with the natural bases, especially T or U, by the electron negative repulsion with the 4-keto group of T or U, as well as the steric clash.
Although the 98% selectivity of the unnatural base pairing is not sufficient for practical use in replication (the retention rate of the isoG-isoC pair in 20-cycle PCR amplified DNA fragments becomes around 67% (0.98 (20) = ~0.67)), this is a unique expansion system for PCR amplification.
We still do not know the mechanism underlying the improved fidelity of the unnatural base pairing using the [gamma]-amidotriphosphates.
"Our model is that during this period when a single-strand region is bowed out, you could have
base pairing between this upstream repeat and this downstream repeat to give you [a loop]," Wallace explains.