Allele-specific enrichment: a method for the detection of low level N-ras gene mutations in acute myeloid leukemia.
Point mutations involving codons 12, 13, and 61 of the N-ras gene are found in patients with acute myeloid leukemia (AML). We have developed a sensitive assay for the analysis of these mutations which we have called allele-specific enrichment. In this protocol the polymerase chain reaction (PCR) amplifies DNA with primers that introduce new restriction sites into the normal N-ras allele only. Digestion with the appropriate enzyme cleaves normal, but not mutant, alleles and this digested product provides a mutant allele-enriched template for a second round of amplification. The second PCR product is digested, Southern blotted and analyzed by allele-specific oligonucleotide (ASO) hybridization. This protocol is more sensitive than ASO hybridization alone and has revealed a minor clone in the DNA of a patient with AML. The method may be useful for the detection of minimal residual disease in a subset of patients in remission.
Todd
A V
AV
Kanematsu Laboratories, Royal Prince Alfred Hospital, Camperdown, NSW, Australia.
Ireland
C M
CM
Iland
H J
HJ
D000595:Amino Acid Sequence; D015139:Blotting, Southern; D011905:Genes, ras; D006801:Humans; D007951:Leukemia, Myeloid / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction
D015345:Oligonucleotide Probes