PCR AMPLIFICATION OF 40-YEAR-OLD PARAFFIN-EMBEDDED TUMOR-TISSUES - COMPARISON OF 4 DIFFERENT DNA EXTRACTION AND PURIFICATION METHODS

Four different methods of DNA extraction from formalin-fixed, paraffin -embedded tissues were compared for their ability to produce DNA suita ble as a template for polymerase chain reaction (PCR). Seven paraffin- embedded rhabdomyosarcoma tissue blocks from the 1950s and one from 19 60 were treated with the following extraction methods: 1. Proteinase K digestion and phenol/chloroform extraction; 2. Proteinase K digestion followed by boiling to inactivate the enzyme; 3. Proteinase K digesti on, addition of Chelex-100, followed by boiling; and 4. Proteinase K d igestion and Prep-A-Gene purification. DNA extracted by methods 1 and 2 was degraded, but DNA of high molecular weight was recovered in ever y sample extracted by methods 3 and 4 - even though some degradation w as observed. Extracted DNA was used as a template for PCR amplificatio n of exon 4 of the PAX-3 gene. PCR was successful in 7 out of 8 sample s prepared using methods 3 and 4, producing levels of amplified produc t equivalent to those obtained with control DNA obtained from fresh ly mphocytes. Only very weak products were found in samples prepared by m ethods 1 (2 out of 8) and 2 (4 out of 8). These results indicate that chelation of polyvalent ions (Chelex-100 method) or obviating the need for boiling (Prep-A-Gene) may protect DNA during extraction.