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 develop ed to be used at the ultrastructural level and with a high degree of r esolution, Samples applied to test the method were fresh cell lines (C aSki, SiHa) and HPV-16 DNA-containing tissues rescued from routine par affin blocks, The specimens were embedded in Epon-Araldite and/or hydr ophilic-resin LRWhite. Ultrathin sections mounted on grids were subjec ted to G-PCR using an HPV-16-specific primer set, The amplified produc ts were identified by auro-immunohistochemical labelling of the biotin ylated nucleotide, The results indicated successful amplification of t arget DNA in both cell and tissue samples, being confined to the intra nuclear region, The negative controls [HeLa cells, isolated mammary ca rcinoma cell cultures (MCF 7, and T47-D) (ATCC) (U.S.A.), normal tyroi d tissue and steroid-producing tumour tissue] failed to exhibit any am plification of the target DNA sequences. The sensitivity of the G-PCR system was evaluated by performing a parallel in situ hybridization (I SH) of serial sections, The signals obtained from G-PCR were more inte nse than those of ISI-I and more informative as to the precise subcell ular localization of amplicons. (C) 1997 John Wiley & Sons, Ltd.