N-ras gene mutations in acute myeloid leukemia: accurate detection by solid-phase minisequencing.
Mutations in the N-ras gene are found in one-third of patients with acute myeloid leukemia. The N-ras mutations could serve as markers for residual cells, if a highly sensitive method for detecting the mutations was available. We applied a new method, solid-phase minisequencing, to analyze bone-marrow cells from 16 patients with acute myeloid leukemia for mutations in codon 12, 13 and 61 of the N-ras gene. In the solid-phase minisequencing technique the mutations are identified by a primer extension reaction, in which a single labelled nucleoside triphosphate is incorporated into an immobilized DNA fragment previously amplified by the polymerase chain reaction. We identified N-ras mutations in 5 of the patients (30%). In one patient, we observed 2 mutations that were shown to be located in different alleles. With the solid-phase minisequencing method, we were able to determine the proportion of mutated cells in the samples. We found that in 4 of the samples only a fraction (7-64%) of the blasts carried an N-ras mutation, and in one sample practically all blast cells were mutated. The method was highly sensitive, allowing us to identify N-ras mutations even when the sample consisted of 99.7% normal cells and only 0.3% mutated blasts.
Syvänen
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AC
National Public Health Institute, Laboratory of Molecular Genetics, Helsinki, Finland.
Söderlund
H
H
Laaksonen
E
E
Bengtström
M
M
Turunen
M
M
Palotie
A
A
D001483:Base Sequence; D011905:Genes, ras; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation