A novel grid polymerase chain reaction (G-PCR) approach at ultrastructural level to detect target DNA in cell cultures and tissues.
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.
Kareem
B N
BN
Department of Pathology, Norwegian Radium Hospital, Oslo, Norway.
Karlsen
F
F
Holm
R
R
Hennig
E M
EM
Suo
Z
Z
Emilsen
E
E
Hellesylt
E
E
Nesland
J M
JM
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*
D004279:DNA, Viral