In situ detection of specific p53 mutations in cultured cells using the amplification refractory mutation system polymerase chain reaction.
Accurate molecular detection of genetic mutations involved in tumorigenesis has been based predominantly on analysis of extracted DNA, but this does not provide detailed information on the location, number, type or clonal distribution of mutated cells and their precise anatomic location and clonal distribution. This study has used a sensitive and specific application of the amplification refractory mutation system (ARMS)-polymerase chain reaction (PCR) in situ, combined with in situ hybridization to localize and identify cells with defined p53 mutations. The ARMS-PCR was performed in situ in SW480 cells in suspension and in cells either cultured or cytospun onto glass slides. Amplified mutant DNA PCR products were detected in SW480 cells using digoxigenin-labeled probes, visually identifying cells harboring specific mutations in the p53 gene. In situ hybridization alone of the mutant cells without the amplification step was negative. Normal human fibroblasts or endothelial cells were refractory to in situ amplification. This reaction was mutation-specific as CEM cells with different p53 mutations reacted negatively. Mutant messenger RNA (mRNA) in tumor cells was also selectively amplified in situ by ARMS-PCR following reverse transcription (RT). This study demonstrates the potential of in situ ARMS-PCR or RT-ARMS-PCR for mutation analysis in situ and could have useful clinical applications.
Low
E O
EO
Oral Pathology and Oral Medicine, University of Sydney, Australia.
Gibbins
J R
JR
Walker
D M
DM
D000483:Alleles; D002469:Cell Separation; D002474:Cell-Free System; D004252:DNA Mutational Analysis / Q000379:methods; D004273:DNA, Neoplasm / Q000032:analysis; D016158:Genes, p53 / Q000235:genetics*; D006801:Humans; D017403:In Situ Hybridization; D009154:Mutation / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D012996:Solutions; D014407:Tumor Cells, Cultured
D004273:DNA, Neoplasm; D012333:RNA, Messenger; D012996:Solutions