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 purification of DNA, amplification of gene fragments in various sizes, and screening for mutations. The DNA was extracted from tissue sections with proteinase K, using various procedures, and purified. The mutation cluster region of the APC gene was amplified with polymerase chain reaction to generate either two large or four small overlapping DNA fragments. The GC-clamped fragments were screened for mutations with temperature gradient gel electrophoresis, and mutations were identified with sequencing. The DNA was easily amplified as large fragments from fresh or unfixed-frozen samples. However, DNA amplification of large fragments from archival samples was successful in 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. Polymerase chain reaction of smaller fragments, with sizes between 200 and 270 base pairs (bp), was successful for 97% of the amplification reactions when using DNA that was purified with silica. Screening with temperature gradient gel electrophoresis was reproducible and sensitive with a detection limit of 5% mutated DNA in the presence of an excess of wild-type DNA.
Centre de Génétique Humaine, Centre Hospitalier Universitaire du Sart Tilman, Université de Liège, Belgium.
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Poncin
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Mulkens
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Arends
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de Goeij
D000230:Adenocarcinoma / Q000235:genetics* / Q000473:pathology; D000236:Adenoma / Q000235:genetics* / Q000473:pathology; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000473:pathology; D004252:DNA Mutational Analysis; D017931:DNA Primers / Q000737:chemistry; D004273:DNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D019286:Endopeptidase K / Q000378:metabolism; D005404:Fixatives; D005557:Formaldehyde; D017491:Genes, APC / Q000235:genetics*; D006801:Humans; D009154:Mutation / Q000235:genetics*; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D004273:DNA, Neoplasm; D005404:Fixatives; D005557:Formaldehyde; D019286:Endopeptidase K