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This deamination process is normally counteracted by uracil-N-glycosylase, the product of the ung gene, and organisms defective in the removal of uracil from DNA have an increased spontaneous mutation rate and more G:C right arrow] A:T base-pair transitions (7).
An effective hot start is provided here by the use of dUTP and uracil-N-glycosylase in the PCR.
To prevent carryover, 200 [micro]M of uracil triphosphate was part of the master mixture, and uracil-N-glycosylase was used systematically.