title: Screening for the beta-39 mutation in thalassemia by capillary electrophoresis in free solution in strongly acidic, isoelectric buffers.
abstract: A novel method is reported for screening for point mutations in genomic DNA: free-zone capillary electrophoresis in very acidic buffers. This method exploits the charge difference among the four different bases (C, T, A, G) in a pH window between 2.5 and 3.5, where the four titration curves fan out. The method is applied to the detection of the beta-39 missense mutation in the beta-globin gene in thalassemias. A 60-mer fragment straddling the mutation site has been amplified. In an isoelectric buffer (iminodiacetic acid) of pH 3.3, partial resolution between the wild type and mutated strands is obtained. In a pH 3.0 phosphate buffer, baseline resolution is achieved between the two strands in a heterozygous individual. Due to the short size of the amplified fragment, this method can only be applied to routine screening for known mutations because resolution was lost in a fragment 100 bases long.
authors.affiliation: CNR, Institute of Advanced Biomedical Technologies, Segrate (Milano), Italy. gelfi@itba.mi.cnr.it
authors.forename: C
authors.initials: C
authors.lastname: Gelfi
authors.forename: A
authors.initials: A
authors.lastname: Viganó
authors.forename: P
authors.initials: P
authors.lastname: Carta
authors.forename: P
authors.initials: P
authors.lastname: Manchia
authors.forename: G F
authors.initials: GF
authors.lastname: Cossu
authors.forename: P G
authors.initials: PG
authors.lastname: Righetti
mesh_terms: D002021:Buffers; D004247:DNA / Q000097:blood; D019075:Electrophoresis, Capillary / Q000379:methods; D005914:Globins / Q000235:genetics*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008403:Mass Screening / Q000379:methods; D020125:Mutation, Missense; D016133:Polymerase Chain Reaction / Q000379:methods; D017086:beta-Thalassemia / Q000097:blood / Q000175:diagnosis* / Q000235:genetics
chemical_list: D002021:Buffers; D005914:Globins; D004247:DNA