RESUMO

In the present work, a comparative study of 5-FdUrd, thy-, and metabolic in vivo labeling methods for plasmid and chromosomal DNA in E. coli DH5alpha cells was performed in order to achieve the best thymidine substitution method by 5-BrdUrd. According to the colorimetric immunoenzymatic results, we found that the minimal detectable labeled DNA (MDLD) was 312pg with the 5-FdUrd and thy- methods for 5-BrdUrd labeled plasmid DNA. 5-BrdUrd replaced about 96% of the total thymidine by 5-FdUrd methods; for the thy- and metabolic labeling methods, the MDLD value was 1,25 ng for denatured 5-BrdUrd chromosomal DNA. Pyrimidine nucleoside analogues were also evaluated as immunochemical markers for their in vivo introduction into DNA.

Assuntos

Bromodesoxiuridina , DNA/análise , Escherichia coli/genética , Floxuridina , Cromossomos Bacterianos/genética , Cromossomos Bacterianos/metabolismo , Colorimetria/métodos , DNA/metabolismo , Inibidores Enzimáticos , Escherichia coli/metabolismo , Técnicas Imunoenzimáticas , Plasmídeos/análise , Plasmídeos/biossíntese , Timidina/química , Timidina/genética , Timidilato Sintase/antagonistas & inibidores , Timidilato Sintase/metabolismo