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 into paraffin or stored in formalin for up to 26 years. Extraction conditions were optimized for proteinase K digestion, i.e., enzyme concentration, digestion temperature and incubation time. Using the polymerase chain reaction (PCR), DNA was successfully amplified from archival material and sequenced employing a direct nonradioactive cycle sequencing protocol. In general, tissue embedded into paraffin following brief fixation in formalin gave good quantitative results, i.e., up to 1 microgram DNA/mg tissue were extracted. This yield was at least one order of magnitude higher than that obtained with tissue stored in formalin. However, paraffin-embedded neuropathological material was found to contain an as-yet-unidentified PCR inhibitor, and a deleterious effect of long-term fixation in unbuffered low-grade formalin was clearly detectable. Importantly, both paraffin-embedded tissue blocks and human brain that had been stored in formalin for many years yielded DNA sufficient for qualitative analysis. The implications of these findings for the use of neuropathological material in molecular genetic studies are discussed.
Kösel
S
S
Laboratory of Molecular Neuropathology, Ludwig-Maximilians-University, Munich, Germany.
Graeber
M B
MB
D000368:Aged; D000369:Aged, 80 and over; D001483:Base Sequence; D001923:Brain Chemistry; D004247:DNA / Q000302:isolation & purification*; D005260:Female; D005557:Formaldehyde; D006801:Humans; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods*
D005557:Formaldehyde; D004247:DNA