5-Bromodeoxyuridine in vivo labelling of M13 DNA, and its use as a non-radioactive probe for hybridization experiments.
We describe the in vivo production of 5-bromodeoxyuridine- (5-BUdR) labelled M13 DNA by a thymine-requiring Escherichia coli strain. We show that the 5-BUdR-labelled M13 single-stranded DNA is not extruded into the culture medium, but accumulates inside the bacterial cells. On the basis of this observation, a procedure involving FPLC gel filtration already reported and used for the isolation of plasmid DNA has been adapted for the isolation of at least 90% pure 5-BUdR-labelled single-stranded DNA. An M13 probe, containing part of the Hepatitis B Virus (HBV) genome was constructed, and the corresponding 5-BUdR-labelled single-stranded DNA was used in hybridization experiments to detect homologous HBV target DNA. Picogram amounts (10(-19) moles) of the probe itself or the target DNA could be detected, by monoclonal anti-5-BUdR antibodies in an immunoenzymatic assay.
Sakamoto
H
H
Laboratoire des Sondes Froides, Institut Pasteur, Paris, France.
Traincard
F
F
Vo-Quang
T
T
Ternynck
T
T
Guesdon
J L
JL
Avrameas
S
S
D000818:Animals; D001973:Bromodeoxyuridine / Q000276:immunology* / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D005819:Genetic Markers; D007150:Immunohistochemistry / Q000379:methods; D007159:Immunologic Tests / Q000379:methods*; D009693:Nucleic Acid Hybridization; D013936:Thymidine / Q000378:metabolism
D004277:DNA, Single-Stranded; D005819:Genetic Markers; D001973:Bromodeoxyuridine; D013936:Thymidine