Enzymic detection of uracil in a cloned and sequenced deoxyribonucleic acid segment.
Uracil can occur in DNA either as a result of utilization of dUTP by DNA polymerases or from in situ deamination of cytosine. The latter results in transition mutations following the next round of replication. We describe a technique for the detection of uracil in DNA by a modification of the Maxam-Gilbert sequencing procedure. Reaction of end-labeled DNA with uracil-DNA glycosylase followed by 1 M piperidine results in scission products corresponding to locations of uracils. These are detected by autoradiography following electrophoresis through a sequencing gel. Comparison of these scission products with the DNA sequences elucidates the mechanism of origin of the DNA uracils. The technique was tested with a cloned human DNA sequence grown in a dut-,ung-strain of Escherichia coli, which incorporates uracil in place of thymine in its DNA, and by chemical deamination of cytosines in that sequence. The technique was expanded by use of alkaline and enzymic probes to investigate possible accumulation of uracil, base losses, and other modifications in human liver and brain DNA. No damaged DNA moieties were detected. This method is applicable to the study of any recoverable reiterated sequence by any enzyme preparation that can recognize modifications in DNA.
D000293:Adolescent; D000368:Aged; D001483:Base Sequence; D002283:Carcinoma, Bronchogenic / Q000378:metabolism; D018288:Carcinoma, Small Cell / Q000378:metabolism; D002531:Cerebellum / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D004273:DNA, Neoplasm / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000378:metabolism; D008297:Male; D010957:Plasmids; D014498:Uracil / Q000032:analysis*
D004273:DNA, Neoplasm; D014498:Uracil; D004247:DNA; D004259:DNA-Directed DNA Polymerase