Analysis of N‐ras gene mutations in acute myeloid leukemia by allele specific restriction analysis

Abstract

N-ras gene activation occurs via single base substitutions in codons 12, 13, and 61. We have developed a rapid screening method, termed allele specific restriction analysis (ASRA), for detection of N-ras mutations at these three critical codons in acute myeloid leukemia (AML). Patient DNA samples are amplified by the polymerase chain reaction (PCR) by using primers that induce restriction sites in normal but not mutant N-ras alleles. We have used ASRA to identify 5 point mutations in four out of 19 patients at initial presentation of de novo AML. Three patients had one mutation at codon 12, 13, or 61 respectively, while a fourth patient had concurrent mutations at codons 12 and 13. N-ras mutations were more common in patients over 65 years of age (P less than 0.04), but did not correlate with FAB classification, attainment of complete remission, disease free survival, or overall survival. ASRA can also be used as the first step in a more sensitive approach to the detection of ras mutations. When ASRA was combined with allele specific oligonucleotide (ASO) hybridization the sensitivity and specificity of these assays were increased. This allowed identification of additional low level mutations in two patients. The data presented here constitute the first complete analysis of N-ras mutations in leukemia by ASRA and include the first identification of three concurrent N-ras mutations in a single leukemic patient. By facilitating sensitive sequential studies, ASRA should contribute to our understanding of the role of N-ras mutations in leukemogenesis.

CASP8 D302H polymorphism delays the age of onset of breast cancer in BRCA1 and BRCA2 carriers

“…The methods used to detect RAD51 and TP53 polymorphisms were based on allele-specific restriction enzyme site analysis (ASRA) [37]. The assessment of RAD51 -135C [ G polymorphism was performed amplifying by PCR the 483 bp region around nucleotide 135 using the primers RAD51F (5 0 -GCAACTCATCTGGGTTGTGC-3 0 ) and RAD51R (5 0 -TCAGGAATCCGGAAGCCCTG-3 0 ) at 1.5 mM MgCl 2 .…”

Alterations in IRF1/IRF2 expression in acute myelogenous leukemia

“…After being dried, the DNA was fixed onto the filter by UV light. The filters were pre-hybridized with 5× SSPE, 5× Denhardts, 0.5% SDS, 100 mM sodium pyrophosphate (pH 7.5), 50°C for 2 hr [4] or 55°C for 2 hr [14,15], and then hybridized with oligonucleotides and labeled with [␥-32 P]ATP and polynucleotide kinase (Amersham) overnight at 50-55°C. This was followed by stringent washing in 2× SSC at room temperature for 30 min, TMAC solution (TMAC 3 mol, EDTA 2 mM, 0.1% SDS) for 1 hr, and a final rinse with 6× SSC.…”

A commented dictionary of techniques for genotyping

“…After locusspecific PCR, amplicons are digested with different allele-specific restrictases. Restriction patterns are detected in the gel and genotypes are determined according to the molecular weight of the fragments observed [118][119][120]. PCR-RFLP can be performed with informative enzymes that have a single recognition site in some alleles but none in other alleles.…”