Efficient detection of point mutations on color-codedstrands of target DNA.

Abstract

Presently available methods for screening large genetic regions for unknown point mutations are neither flawless nor particularly efficient. We describe an approach, especially well suited to identiyng mutations present in the heterozygous state, that combines several improvements in a protocol called fluorescence-assisted mmath analysis (FAMA). Appropriate gene regions of the wild-type and the putative mutant allele are simultaneously amplified from genomic DNA by using the polymerase chain reaction, and large DNA fragments, so far up to 800 bp, are end labeled with strand-specific fluorophores. Aliquots are denatured and reannealed to form heteroduplexes and subjected to conventional cytosne-and thymine-specific modfifications. Cleavages occuring on opposite strands are detected by denatring gel electrophoresis using an automated DNA sequencer. Since the DNA a nts derived from the mutant allele are also end labeled, the number of informative mispaired bases is doubled compared to conventional searches using wild-type probes. The sensiivty of detection is also increased, because differential fluorescent end labeling allows the identification and measurement ofstand-specific background cleavages at matched cytosine or thymine residues. Automatic superimposition of tracings from different subjects allows mia detection at sites that, because of the nature of the bases involved and of the neighboring sequence, are known to be less susceptible to cleavage. The effects of the latter parameters have been studied quantitatively on a series of point mutations found in the human Cl-inhibitor gene in patents affected by hereditary angioedema. Dilution experiments have demonstrated that most mutations are detected even when the mutant chromosome is diluted 10-fold or more compared with the normal one.The identification of loci responsible for a large number of monogenic diseases and of a growing number of genes involved in tumor predisposition and multifactorial diseases has rapidly expanded the range of application of DNA diagnosis. Since the majority of inherited disorders are heterogeneous at the gene level and are more often due to subtle changes affecting one or several nucleotides than to large deletions or duplications, considerable efforts have been made to develop methods able to detect not only mutations previously identified in probands or known to be prevalent in a given population but also as yet unknown mutations (for reviews, see refs. 1-3). Each of the methods has, however, disadvantages that render its use problematic when searching unknown point mutations within large DNA regions. Denaturing gradient gel electrophoresis (4-6) requires, for example, computer-assisted optimization of the target region and of the electrophoresis conditions, depending on the sequence content of the DNA fragment. The single-strand conformation polymorphism technique (7), in spite of its experimental simplicity, has shown limited sensitivity for the detection of mutations in DNA fragments longer than 200 bp (8, 9).Neith...

Cellular responses to ionising radiation of AT heterozygotes: differences between missense and truncating mutation carriers

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Multiple molecular mechanisms contribute to radiation sensitivity in mantle cell lymphoma

“…To determine the molecular status of ATM in our cell lines, fluorescence-assisted mismatch analysis (FAMA) was chosen as the most appropriate method for mutation detection (Verpy et al, 1994). The entire coding region of ATM gene was examined in both MCL cell lines.…”

Phenotypic cellular characterization of an Ataxia telangiectasia patient carrying a causal homozygous missense mutation

“…The ATM cDNA was used as a template for PCR and then for Fluorescence-Assisted Mismatch Analysis (FAMA) (Verpy et al, 1994). ATM exons 4, 5, 6, 11 and 65 were analyzed by direct sequencing using the Big Dye Terminator v2.0 cycle sequencing kit (Applied Biosystem).…”