Comparative study of three methods for non-radioactive in vivo DNA labeling in Escherichia coli using nucleoside analogs.
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.
Pérez-Bello
Dannelys
D
Molecular Biology Department, Neurosciences Center, Havana, Cuba.
Suárez
Viana Manrique
VM
Bolaños
Celso Pérez
CP
López
Judith Miranda
JM
Pérez
Gabriel Pérez
GP
López-Canovas
Lilia
L
Higginson-Clarke
David
D
Rodríguez-Tanty
Chryslaine
C
Riverón
Ana María
AM
D001973:Bromodeoxyuridine; D002876:Chromosomes, Bacterial / Q000235:genetics / Q000378:metabolism; D003124:Colorimetry / Q000379:methods; D004247:DNA / Q000032:analysis* / Q000378:metabolism; D004791:Enzyme Inhibitors; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005467:Floxuridine; D007124:Immunoenzyme Techniques; D010957:Plasmids / Q000032:analysis / Q000096:biosynthesis; D013936:Thymidine / Q000737:chemistry / Q000235:genetics; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism
D004791:Enzyme Inhibitors; D005467:Floxuridine; D004247:DNA; D013940:Thymidylate Synthase; D001973:Bromodeoxyuridine; D013936:Thymidine