Molecular methods for the detection of mutations

Abstract

We report the results of a collaborative study aimed at developing reliable, direct assays for mutation in human cells. The project used common lymphoblastoid cell lines, both with and without mutagen treatment, as a shared resource to validate the development of new molecular methods for the detection of low-level mutations in the presence of a large excess of normal alleles. As the "gold standard, " hprt mutation frequencies were also measured on the same samples. The methods under development included i) the restriction site mutation (RSM) assay, in which mutations lead to the destruction of a restriction site; ii) minisatellite length-change mutation, in which mutations lead to alleles containing new numbers of tandem repeat units; iii) loss of heterozygosity for HLA epitopes, in which antibodies can be used to direct selection for mutant cells; iv) multiple fluorescence-based long linker arm nucleotides assay (mf-LLA) technology, for the detection of substitutional mutations; v) detection of alterations in the TP53 locus using a (CA) array as the target for the screening; and vi) PCR analysis of lymphocytes for the presence of the BCL2 t(14:18) translocation. The relative merits of these molecular methods are discussed, and a comparison made with more "traditional" methods.

Frequent HPRT mutations in paroxysmal nocturnal haemoglobinuria reflect T cell clonal expansion, not genomic instability

“…Analysis of HPRT mutations, which confer a selective advantage for cells in culture, has been utilized for this purpose (Monteiro et al , 2000; Albertini, 2001). An increase in mutant frequency (Mf) detected by HPRT has been linked to environmental exposures to mutagens such as smoking, malignancy and in cancer postchemotherapy (Abramson et al , 1999; Monteiro et al , 2000; Albertini, 2001; Rice et al , 2003). However, the assay is dependent on the proliferation of lymphocytes, mainly T cells, in the culture system.…”