Screening for the beta-39 mutation in thalassemia by capillary electrophoresis in free solution in strongly acidic, isoelectric buffers |
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Authors: | Gelfi C Viganó A Carta P Manchia P Cossu G F Righetti P G |
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Institution: | CNR, Institute of Advanced Biomedical Technologies, Segrate (Milano), Italy. gelfi@itba.mi.cnr.it |
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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. |
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