A novel grid polymerase chain reaction (G-PCR) approach at ultrastructural level to detect target DNA in cell cultures and tissues
✍ Scribed by Kareem, Bilquis N.; Karlsen, Frank; Holm, Ruth; Hennig, Elin M.; Suo, Zhenhe; Emilsen, Elisabeth; Hellesylt, Ellen; Nesland, Jahn M.
- Publisher
- John Wiley and Sons
- Year
- 1997
- Tongue
- English
- Weight
- 609 KB
- Volume
- 183
- Category
- Article
- ISSN
- 0022-3417
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✦ Synopsis
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. 1997 John Wiley & Sons, Ltd.