Optimizing the APC gene mutation analysis in archival colorectal tumor tissue

Critical steps in the methodology of mutation analysis on routinely fixed, paraffin-embedded samples have been evaluated, including extraction and pur ification of DNA, amplification of gene fragments in Various sizes, and scr eening for mutations. The DNA was extracted from tissue sections with prote inase K, using various procedures, and purified. The mutation cluster regio n of the APC gene was amplified with polymerase chain reaction to generate either two large or four small overlapping DNA fragments. The GC-clamped fr agments were screened for mutations with temperature gradient gel electroph oresis, and mutations were identified with sequencing. The DNA was easily a mplified as large fragments from fresh or unfixed-frozen samples. However, DNA amplification of large fragments from archival samples was successful i n only 40 of the 114 tumor specimens analyzed (35%). Prolonged extraction, either at 55 degrees C or at 37 degrees C, gave no better results. Polymera se chain reaction of smaller fragments, with sizes between 200 and 270 base Fairs (bp), was successful for 97% of the amplification reactions when usi ng DNA that was purified with silica. Screening with temperature gradient g el electrophoresis was reproducible and sensitive with a detection limit of 5% mutated DNA in the presence of an excess of wild-type DNA.