title: A novel grid polymerase chain reaction (G-PCR) approach at ultrastructural level to detect target DNA in cell cultures and tissues.
abstract: A novel grid polymerase chain reaction (G-PCR) method has been developed to be used at the ultrastructural level and with a high degree of resolution. Samples applied to test the method were fresh cell lines (CaSki, SiHa) and HPV-16 DNA-containing tissues rescued from routine paraffin blocks. The specimens were embedded in Epon-Araldite and/or hydrophilic-resin LRWhite. Ultrathin sections mounted on grids were subjected to G-PCR using an HPV-16-specific primer set. The amplified products were identified by auro-immunohistochemical labelling of the biotinylated nucleotide. The results indicated successful amplification of target DNA in both cell and tissue samples, being confined to the intranuclear region. The negative controls [HeLa cells, isolated mammary carcinoma cell cultures (MCF 7, and T47-D) (ATCC) (U.S.A.), normal thyroid tissue and steroid-producing tumour tissue] failed to exhibit any amplification of the target DNA sequences. The sensitivity of the G-PCR system was evaluated by performing a parallel in situ hybridization (ISH) of serial sections. The signals obtained from G-PCR were more intense than those of ISH and more informative as to the precise subcellular localization of amplicons.
authors.affiliation: Department of Pathology, Norwegian Radium Hospital, Oslo, Norway.
authors.forename: B N
authors.initials: BN
authors.lastname: Kareem
authors.forename: F
authors.initials: F
authors.lastname: Karlsen
authors.forename: R
authors.initials: R
authors.lastname: Holm
authors.forename: E M
authors.initials: EM
authors.lastname: Hennig
authors.forename: Z
authors.initials: Z
authors.lastname: Suo
authors.forename: E
authors.initials: E
authors.lastname: Emilsen
authors.forename: E
authors.initials: E
authors.lastname: Hellesylt
authors.forename: J M
authors.initials: JM
authors.lastname: Nesland
mesh_terms: D002460:Cell Line; D004279:DNA, Viral / Q000032:analysis*; D005260:Female; D006801:Humans; D017403:In Situ Hybridization; D008854:Microscopy, Electron; D027383:Papillomaviridae / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D002583:Uterine Cervical Neoplasms / Q000648:ultrastructure / Q000821:virology*
chemical_list: D004279:DNA, Viral