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Screening for the β‐39 mutation in thalassemia by capillary electrophoresis in free solution in strongly acidic, isoelectric buffers
Author(s) -
Gelfi Cecilia,
Viganó Agnese,
Carta Piera,
Manchia Pierangela,
Cossu Gian Franco,
Righetti Pier Giorgio
Publication year - 2000
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/(sici)1522-2683(20000301)21:4<780::aid-elps780>3.0.co;2-y
Subject(s) - capillary electrophoresis , isoelectric focusing , chemistry , microbiology and biotechnology , point mutation , iminodiacetic acid , electrophoresis , isoelectric point , resolution (logic) , mutation , chromatography , phosphate buffered saline , missense mutation , biochemistry , biology , gene , chelation , enzyme , artificial intelligence , computer science , organic chemistry
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 β‐39 missense mutation in the β‐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.