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Human papillomavirus in cervix carcinoma and condylomata acuminate—identification of HPV‐DNA by improved dot‐blot‐hybridization
Author(s) -
KNOBLER R.M.,
SCHNEIDER S.,
RADLWIMMER B.,
BODEMER W.,
GEBHART W.,
SÖLTZSZÖTS J.
Publication year - 1992
Publication title -
clinical and experimental dermatology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.587
H-Index - 78
eISSN - 1365-2230
pISSN - 0307-6938
DOI - 10.1111/j.1365-2230.1992.tb00245.x
Subject(s) - dot blot , human papillomavirus , virology , cervix , dna–dna hybridization , southern blot , dna , in situ hybridization , biology , pathology , medicine , cancer , gene , genetics , gene expression
Summary Using the benzoylated naphthoylated DEAE cellulose method (BND‐method) we have designed a more efficient approach for the detection of human papillomavirus‐DNA (HPV‐DNA) via dot‐blot and hybridization. Biopsy material from anogenital warts (40 patients), invasive carcinoma uteri (12 patients) and normal controls (20 patients) were studied for the presence of HPV‐DNA. Phenol extracted DNA from representative lesions was loaded onto a pretreated nitrocellulose filter, was incubated under stringent conditions with 32 ‐P‐dCTP labelled HPV‐DNA and exposed to a Kodak X‐OMAT film. DNA of HPV types 11, 16, 18 were cloned into plasmid vectors. The common, time‐consuming caesium‐chloride density‐gradient centrifugation used for purification of plasmid DNA (20–36 h), was substituted by the BND‐method (15 min). Complete HPV genomes were excised using the restriction endonucleases Eco RI and Bam HI. The HPV‐DNA fragments obtained were then electroeluted using the ‘Bio‐Trap’ method and subsequently labelled with 32 P‐dCTP by nick translation. Without resorting to more complex and sensitive technology, such as the polymerase chain reaction, efficiency of specific analysis of large numbers of cervical samples and condylomata was achieved without loss of accuracy or increased costs. The time required for HPV identification from biopsy or sample receipt was shortened considerably (approximately 50%).