Molecular methods for the detection of mutations

• Published in: Teratogenesis, carcinogenesis, and mutagenesis Vol. 20; no. 6; pp. 357 - 386
• Main Authors: Monteiro, C., Marcelino, L.A., Conde, A.R., Saraiva, C., Giphart-Gassler, M., De Nooij-van Dalen, A.G., Van Buuren-van Seggelen, V., Van der Keur, M., May, C.A., Cole, J., Lehmann, A.R., Steinsgrimsdottir, H., Beare, D., Capulas, E., Armour, J.A.L.
• Format: Journal Article
• Language: English
• Published: New York John Wiley & Sons, Inc 2000; Wiley-Liss
• Subjects: Base Sequence; BCL2 (AR Lehmann); Biological and medical sciences; Cell Line; DNA Mutational Analysis - methods; Fluorescent Dyes; Fundamental and applied biological sciences. Psychology; Genes, p53; HLA locus; HLA-A Antigens - genetics; hprt; human population monitoring; Humans; Hypoxanthine Phosphoribosyltransferase - genetics; instability; Loss of Heterozygosity; Lymphocytes - drug effects; Lymphocytes - radiation effects; mf-LLA; minisatellite; Minisatellite Repeats; minisatellites; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; mutagenesis (J Cole); Mutagenesis. Repair; Mutagenicity Tests - methods; Mutation; p53; Point Mutation; Proto-Oncogene Proteins c-bcl-2 - genetics; Restriction Mapping; restriction site mutations; Sensitivity and Specificity; small-pool PCR (CA May); Translocation, Genetic; Chromosomal aberration; Human; HLA-System; Methodology; Restriction fragment; Selectivity; Review; Mutagen; In vitro; TP53 Gene; Mutagenesis; Investigation method; Minisatellite DNA; DNA; Established cell line; Point mutation; Loss of heterozygosity; Abnormal chromosome; Mutation; Detection; Onc gene; Lymphoblastoid cell; Tumor suppressor gene; Chromosome translocation
• ISSN: 0270-3211; 1520-6866
• DOI: 10.1002/1520-6866(2000)20:6<357::AID-TCM5>3.0.CO;2-G

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. Teratogenesis Carcinog. Mutagen. 20:357–386, 2000. © 2000 Wiley‐Liss, Inc.