title: Detection of single-nucleotide mutations including substitutions and deletions by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
abstract: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was applied to the detection of mutations involving single nucleotides. For detection of a single-nucleotide deletion, a normally 44 bp region of the L1CAM gene was amplified in a 50 microL solution, and measurement was carried out on the DNA sample after phenol extraction and ethanol precipitation. A molecular mass decrease of 300 Da corresponding to a single nucleotide was identified in the amplified product of patient DNA. For detection of a substitution, an amplified product from a 50 bp region of the human beta-globin gene was cleaved with restriction endonucleases HaeIII and Bsp12861. Measurement of a mixture of digested fragments, or restriction fragment mass mapping, clearly identified a heterozygous G/C mutation in the molecular ion signals for both sense and antisense single-stranded DNAs. The results indicate that MALTI-TOFMS is feasible for genetic diagnosis of point mutations.
authors.affiliation: Department of Molecular Medicine, Osaka Medical Center, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Wada
authors.forename: M
authors.initials: M
authors.lastname: Yamamoto
mesh_terms: D004247:DNA / Q000032:analysis* / Q000235:genetics; D017353:Gene Deletion; D005914:Globins / Q000235:genetics; D006801:Humans; D009154:Mutation; D009711:Nucleotides / Q000032:analysis*; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D009711:Nucleotides; D005914:Globins; D004247:DNA