Enrichment of mutant alleles by chromatographic removal of wild type alleles: a new principle for the detection of alleles with unknown point mutations at excess of wild type alleles.
In human carcinomas, mutations that alter tumour genes such as the KRAS, P53, or APC genes, are mostly point mutations. The detection of mutant alleles of tumour genes in specimens such as urine, pancreatic juice, sputum, and stool holds great promise for an early diagnosis of cancer. In addition, the detection of mutant tumour genes in tissue samples, such as lymph nodes or resection margins, may allow a sensitive diagnosis of residual malignant disease. However, the reliable detection of mutant alleles in excess of wild type alleles remains an unresolved analytical problem when the mutations are not known a priori. In the present communication, a new approach is described which makes possible the detection of unknown point mutations in tumour genes at excess of wild type alleles. The method is based on the removal of wild type alleles by hybridisation to immobilised complementary oligonucleotides. Using this approach, an enrichment of mutant KRAS, P53 and APC alleles of one mutant in up to 10(3) normal alleles has been achieved. Parallel miniaturised separation units with oligonucleotides complementary to defined sequences of a wild type allele should allow the detection of unknown point mutations as well as small insertions or deletions which occur in the sequence range covered by the oligonucleotides.
Nollau
P
P
Abteilung für Klinische Chemie, Medizinische Klinik, Universitätskrankenhaus Eppendorf, Hamburg, Germany.
Fischer
C
C
Tschentscher
P
P
Wagener
C
C
D000483:Alleles; D002845:Chromatography / Q000379:methods*; D004273:DNA, Neoplasm / Q000737:chemistry*; D006801:Humans; D017354:Point Mutation; D012150:Polymorphism, Restriction Fragment Length; D017422:Sequence Analysis, DNA; D014407:Tumor Cells, Cultured
D004273:DNA, Neoplasm