A novel grid polymerase chain reaction (G-PCR) approach at ultrastructural level to detect target DNA in cell cultures and tissues

作者：Kareem BN; Karlsen F; Holm R; Hennig EM; Suo ZH; Emilsen E; Hellesylt E; Nesland JM

来源：Journal of Pathology, 1997, 183(4): 486-493.

• PCR
• ultrastructural level
• electron microscopy
• HPV

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 tyroid 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 ISI-I and more informative as to the precise subcellular localization of amplicons.

• 出版日期1997-12