USE OF NEUROPATHOLOGICAL TISSUE FOR MOLECULAR-GENETIC STUDIES - PARAMETERS AFFECTING DNA EXTRACTION AND POLYMERASE CHAIN-REACTION

Nuclear and mitochondrial DNA were extracted from gray matter of human cerebral cortex which had either been formalin-fixed and embedded int o paraffin or stored in formalin for up to 26 years. Extraction condit ions were optimized for proteinase K digestion, i.e., enzyme concentra tion, digestion temperature and incubation time. Using the polymerase chain reaction (PCR), DNA was successfully amplified from archival mat erial and sequenced employing a direct nonradioactive cycle sequencing protocol. In general, tissue embedded into paraffin following brief f ixation in formalin gave good quantitative results, i.e., up to 1 mu g DNA/mg tissue were extracted. This yield was at least one order of ma gnitude higher than that obtained with tissue stored in formalin. Howe ver, paraffin-embedded neuropathological material was found to contain an as-yet-unidentified PCR inhibitor, and a deleterious effect of lon g-term fixation in unbuffered low-grade formalin was clearly detectabl e. Importantly, both paraffin-embedded tissue blocks and human brain t hat had been stored in formalin for many years yielded DNA sufficient for qualitative analysis. The implications of these findings for the u se of neuropathological material in molecular genetic studies are disc ussed.