title: PCR amplification of 40-year-old paraffin-embedded tumor-tissues - comparison of 4 different DNA extraction and purification methods.
abstract: Four different methods of DNA extraction from formalin-fixed, paraffin-embedded tissues were compared for their ability to produce DNA suitable as a template for polymerase chain reaction (PCR). Seven paraffin-embedded rhabdomyosarcoma tissue blocks from the 1950s and one from 1960 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 digestion, addition of Chelex-100, followed by boiling; and 4. Proteinase K digestion and Prep-A-Gene purification. DNA extracted by methods 1 and 2 was degraded, but DNA of high molecular weight was recovered in every sample extracted by methods 3 and 4 - even though some degradation was observed. Extracted DNA was used as a template for PCR amplification of exon 4 of the PAX-3 gene. PCR was successful in 7 out of 8 samples prepared using methods 3 and 4, producing levels of amplified product equivalent to those obtained with control DNA obtained from fresh lymphocytes. Only very weak products were found in samples prepared by methods 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.
authors.affiliation: MANCHESTER METROPOLITAN UNIV,DEPT BIOL SCI,MANCHESTER M1 5GD,LANCS,ENGLAND. ROYAL MANCHESTER CHILDRENS HOSP,REG MOLEC GENET LAB,MANCHESTER M27 4HA,LANCS,ENGLAND. CHRISTIE HOSP & HOLT RADIUM INST,CLIN RES LABS,TUMOR BIOL LAB,MANCHESTER,LANCS,ENGLAND.
authors.forename: W
authors.initials: W
authors.lastname: Wang
authors.forename: P
authors.initials: P
authors.lastname: Kumar
authors.forename: M
authors.initials: M
authors.lastname: Schwarz
authors.forename: G
authors.initials: G
authors.lastname: Malone
authors.forename: A
authors.initials: A
authors.lastname: Haworth
authors.forename: S
authors.initials: S
authors.lastname: Kumar