Non-isotopic labeling of DNA by newly developed hapten-containing platinum compounds.
A newly developed reagent was tested for its applicability in in situ hybridization and in reversed hybridization of DNA fragments generated by PCR amplification. This Platinum-complex, designated universal linkage system (ULS), equipped, for instance, with biotin or fluorescein as hapten, enables versatile nonenzymatic "one step" labeling of genomic, cloned or amplified DNA. Here we demonstrate direct in situ detection of integrated human papilloma virus (HPV) DNA in cervical carcinoma cells using DNA probes labeled with fluorescein-ULS. In cervical smears the presence of HPV or Chlamydia trachomatis was assessed by PCR. To analyze the amplified DNA, a reversed hybridization assay was developed. Immobilized probes were incubated with amplimers that were labeled post-amplification through the action of the biotinylated (BIO)-ULS complex. This novel type of nonradioactive analysis appeared to be as sensitive as its isotopic or colorimetric equivalents. This labeling procedure is simple, versatile and can be included as a universal hapten linkage system in any PCR test or in situ hybridization assay aiming at the detection and identification of DNA or RNA molecules.
van Belkum
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A
Diagnostic Center SSDZ, Delft, The Netherlands.
Linkels
E
E
Jelsma
T
T
van den Berg
F M
FM
Quint
W
W
D001710:Biotin / Q000031:analogs & derivatives* / Q000378:metabolism; D002692:Chlamydia trachomatis / Q000235:genetics*; D004279:DNA, Viral / Q000032:analysis*; D005260:Female; D006241:Haptens; D006801:Humans; D017403:In Situ Hybridization; D009944:Organoplatinum Compounds / Q000378:metabolism*; D027383:Papillomaviridae / Q000235:genetics*; D010984:Platinum / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D014626:Vaginal Smears
D004279:DNA, Viral; D006241:Haptens; D009944:Organoplatinum Compounds; C086490:biotinylated-ULS platinum complex; D010984:Platinum; D001710:Biotin