Detection of low-frequency mutations in exon 8 of the TP53 gene by constant denaturant capillary electrophoresis (CDCE).
We extended our development of the means to measure point mutations at the DNA level to the problem of detecting TP53 gene variants in the area around tumors where mutant fractions are expected to be as low as one mutant per 1000 wild-type (WT) sequences. We met this criterion by using the following methods. The TP53 exon 8 sequence was amplified from genomic DNA samples under conditions of high polymerase fidelity using a fluorescein-labeled primer. This mixture of WT and mutant sequences was converted to heteroduplexes of mutant and WT sequences by melting and re-annealing. The mixture was resolved by capillary gel electrophoresis under appropriate constant denaturing conditions. Using laser-induced fluorescence, we found that fractions as low as 1/1000 could be detected without any prior enrichment for mutant sequences, and that the melting properties of the amplified DNA will leave "fingerprints" in the electropherogram that can be used to identify the specific mutation. This method is fast and robust and should be applicable to clinical analyses in which rapid scanning for any mutation in an exonic sequence is important.
Massachusetts Institute of Technology, Cambridge, USA.
P O
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Ekstrøm
A L
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Børresen-Dale
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Qvist
K E
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Giercksky
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WG
Thilly
D003110:Colonic Neoplasms / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods*; D005091:Exons / Q000235:genetics; D005453:Fluorescence; D016158:Genes, p53 / Q000235:genetics*; D020180:Heteroduplex Analysis; D006801:Humans; D009154:Mutation / Q000235:genetics*; D009363:Neoplasm Proteins / Q000235:genetics; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
D009363:Neoplasm Proteins