Detection of N-ras Mutations in Acute Myeloid Leukemia
N-ras gene activation due to single base substitutions has been found in 20–40% of patients with acute myeloid leukemia (AML) depending on the technique used. Differential hybridization to allele specific oligonucleotide (ASO) probes detects mutations in about 22% of patients (1–6) whereas leukemic DNA assayed by NIH/3T3 transfection ± in vivo transformation in nude mice is associated with a mutation frequency of approximately 40% (7–11). We have developed a rapid screening method, termed allele specific restriction analysis (ASRA), for analysis of mutations at codons 12, 13 and 61 of the N-ras gene (12). A similar approach for the identification of Kirsten-ras gene mutations at codon 12 has also been reported (13,14). ASRA involves PCR amplification of DNA or RNA using a mismatched primer which introduces appropriately positioned base substitutions in N-ras and creates a restriction site provided the adjacent sequence is normal. Resistance of the amplified product to digestion indicates the presence of a mutation in the original template.
KeywordsAcute Myeloid Leukemia Acute Myeloid Leukemia Patient K562 Cell Line Single Base Substitution Allele Specific Oligonucleotide
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