Volume 2 Supplement 1
Promising method for DNA extraction from paraffin embedded archive material
© Current Science Ltd 2000
Published: 12 March 2000
Formalin-fixed, paraffin-embedded materials are an invaluable source for mutation analysis. However, DNA extraction from this type of material can be challenging as well as time consuming. DNA extracted from paraffin-embedded material is often highly fragmented and contaminated by protein agents. For DNA analysis, such as PCR (polymerase chain reaction), subsequent TTGE (temporal temperature gradient electrophoresis) and DNA sequencing, optimal conditions require long DNA fragments (200-1000 bp) and a DNA with high purity with an OD ratio (abs. at 260 nm/abs at 280 nm) between 1.6 and 2.0. We have evaluated and combined different protocols to get the highest quality and yield of DNA. 5-10 mm × 8-10 sections of tissue were used.
The best results were achieved from extractions using relatively high volumes (8-10 ml) of xylene and ethanol for the deparaffinization and rehydration steps initiating the extraction protocol. Furthermore, limiting the incubation period for proteinase K digestion of the material to 4-8 hours yields longer fragments of DNA than prolonged digestion. This, however, demands a prolonged incubation period with lysis buffer, up to 24 hours, previous to digestion. The phenol/chloroform extraction step in the traditional extraction procedure contains several uncertain elements, risking protein contamination from the inter-phase between the aqueous and organic phases, and the phenol health hazards are also considerable. Using a PLG (phase lock gel) tube from Eppendorf, in which a gel plug separates the organic phase and the aqueous phase, greatly eases the extraction and increases DNA yield and purity. The organic phase is locked underneath the gel, leaving no room for protein contamination when pipetting off or decanting the upper, aqueous phase. The health risk posed by the solvent vapour released during the isolation of the aqueous phase is also minimised by the gel barrier. Subsequent salt precipitation with 1 M NaCl and ethanol 'rinse' is performed before the samples are air-dried and diluted in 100-200 μ l 1 × TE buffer. DNA yield and quality were evaluated by a spectrophotometer, a fluorometer and PCR fragments separated on an agarose gel followed by EtBr staining. The OD ratio 260/280 nm of the extracted DNA was 1.67-1.97 for different batches. Six out of 10 samples yielded PCR products with fragments as long as 770 bp. A multiplex PCR for 6 exons of the ATM gene was performed with success. Previously extracted DNA from the same type of tissue block using different protocols yielded no PCR products for the same multiplex PCR.
This reliable method of extraction, although a bit time consuming, makes analysis of paraffin-embedded material possible, yielding satisfactory results for further study of the DNA. This protocol will now be used for detection of ATM mutation carriers among family members of AT children who have died of cancer.