Measurement of the sequence specificity of covalent DNA modification by antineoplastic agents using Taq DNA polymerase.
A polymerase stop assay has been developed to determine the DNA nucleotide sequence specificity of covalent modification by antineoplastic agents using the thermostable DNA polymerase from Thermus aquaticus and synthetic labelled primers. The products of linear amplification are run on sequencing gels to reveal the sites of covalent drug binding. The method has been studied in detail for a number of agents including nitrogen mustards, platinum analogues and mitomycin C, and the sequence specificities obtained accord with those obtained by other procedures. The assay is advantageous in that it is not limited to a single type of DNA lesion (as in the piperidine cleavage assay for guanine-N7 alkylation), does not require a strand breakage step, and is more sensitive than other primer extension procedures which have only one cycle of polymerization. In particular the method has considerable potential for examining the sequence selectivity of damage and repair in single copy gene sequences in genomic DNA from cells.
Ponti
M
M
Department of Oncology, University College and Middlesex School of Medicine, London, UK.
Forrow
S M
SM
Souhami
R L
RL
D'Incalci
M
M
Hartley
J A
JA
D000970:Antineoplastic Agents / Q000378:metabolism / Q000494:pharmacology*; D001345:Autoradiography; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000187:drug effects* / Q000378:metabolism; D004249:DNA Damage; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D005784:Gene Amplification; D008969:Molecular Sequence Data; D019914:Taq Polymerase
D000970:Antineoplastic Agents; D004247:DNA; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase