In VivoLabeling of DNA Probes with 5-BrdU

Abstract

The thymidine analogue 5-bromodeoxyuridine (BrdU) can specifically and quantitatively replace the thymidine residue in DNA molecules using eitherin vivo or in vitroenzymatic reaction. BrdU is often used as a substitute for [³Hlthymidine in cytochemistry studies. Indeed, the BrdU labeling of DNA have been used since the 1970s as tools for measuring DNA synthesis in isolated chromosomes and in cells and tissues (Dolbeare, 1995). BrdU is also used to prepare non-radioactive DNA probes in hybridization experiments. These BrdU-labeled probes can be prepared using eitherin vitro or in vivoenzymatic reaction.in vitroBrdU labeling of DNA is performed by nick-translation (Traincard et al., 1983; Niedobitek et al., 1989) or by complementary strand synthesis from recombinant M13 DNA (Frommer et al., 1988).In vivoincorporation of BrdU was proposed to produce BrdU-labeled plasmid (Kitazawa et al., 1989) or single-stranded M13 DNA (Sakamoto et al., 1987). In order to achieve the bestIn vivosubstitution of thymine by 5-bromouracil into the nucleic acid of the phage M13, a thymine-requiring strain ofEscherichia coliwas developed (Sakamoto et al., 1987). With this labeling system, BrdU-labeled M13 single-stranded DNA accumulates inside the bacterial cells, and the alkaline lysis method followed by phenol chloroform extraction (Sambrook et al., 1990) is used to isolate the BrdU-labeled DNA probe. In addition, BrdU end-labeled oligonucleotides can be synthetized (Scippo et al., 1989; Jirikowski et al., 1989).