title: 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.
authors.affiliation: Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Lisboa, Portugal. cm@ihmt.unl.pt
authors.forename: C
authors.initials: C
authors.lastname: Monteiro
authors.forename: L A
authors.initials: LA
authors.lastname: Marcelino
authors.forename: A R
authors.initials: AR
authors.lastname: Conde
authors.forename: C
authors.initials: C
authors.lastname: Saraiva
authors.forename: M
authors.initials: M
authors.lastname: Giphart-Gassler
authors.forename: A G
authors.initials: AG
authors.lastname: De Nooij-van Dalen
authors.forename: V
authors.initials: V
authors.lastname: Van Buuren-van Seggelen
authors.forename: M
authors.initials: M
authors.lastname: Van der Keur
authors.forename: C A
authors.initials: CA
authors.lastname: May
authors.forename: J
authors.initials: J
authors.lastname: Cole
authors.forename: A R
authors.initials: AR
authors.lastname: Lehmann
authors.forename: H
authors.initials: H
authors.lastname: Steinsgrimsdottir
authors.forename: D
authors.initials: D
authors.lastname: Beare
authors.forename: E
authors.initials: E
authors.lastname: Capulas
authors.forename: J A
authors.initials: JA
authors.lastname: Armour
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D004252:DNA Mutational Analysis / Q000379:methods*; D005456:Fluorescent Dyes; D016158:Genes, p53; D015234:HLA-A Antigens / Q000235:genetics; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D019656:Loss of Heterozygosity; D008214:Lymphocytes / Q000187:drug effects / Q000528:radiation effects; D018598:Minisatellite Repeats; D008969:Molecular Sequence Data; D009152:Mutagenicity Tests / Q000379:methods*; D009154:Mutation; D017354:Point Mutation; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics; D015183:Restriction Mapping; D012680:Sensitivity and Specificity; D014178:Translocation, Genetic
chemical_list: D005456:Fluorescent Dyes; D015234:HLA-A Antigens; D019253:Proto-Oncogene Proteins c-bcl-2; D007041:Hypoxanthine Phosphoribosyltransferase