5-Bromo-2′-deoxyuridine triphosphate (Br-dUTP) and dTTP are used interchangeably for DNA synthesis in vitro by the Klenow fragment of Escherichia coli DNA polymerase I. When DNA containing Br-dUMP instead of dTMP at a few preselected sites is transfected into competent bacteria, no mutation occurs, indicating that in vivo E. coli DNA polymerase always places a dAMP residue in front of any unrepaired Br-dUMP residue. On the other hand, in vitro Br-dUTP can also replace dCTP, but only with difficulty: when dCTP is absent, Br-dUMP can be forced in front of a dGMP residue, but the Klenow polymerase pauses before and after addition of Br-dUMP. Transfection into E. coli of the substituted DNA leads to the expected G→A transitions. These mutations can easily be targeted by using a suitable primer and the correctly chosen mix of deoxynucleoside triphosphates containing Br-dUTP. When Br-dUMP has been placed in front of a dGMP residue, the mutation yield is not 100%, showing a partial repair of the transfected DNA before it is replicated. Advantage can be taken of this partial repair to prepare a set of different mutations within a target region in a single experiment.
Pairing properties of bromouracil and repair of bromouracil-containing DNA. Possible utilization of bromodeoxyuridine triphosphate for site-directed mutagenesis
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M Muller, J Martial, W G Verly; Pairing properties of bromouracil and repair of bromouracil-containing DNA. Possible utilization of bromodeoxyuridine triphosphate for site-directed mutagenesis. Biochem J 1 August 1988; 253 (3): 637–643. doi: https://doi.org/10.1042/bj2530637
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