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Capillary electrophoresis combining three‐step multiplex polymerase chain reactions for diagnosing α‐thalassemia
Author(s) -
Chen YenLing
Publication year - 2011
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/elps.201000448
Subject(s) - capillary electrophoresis , multiplex polymerase chain reaction , polymerase chain reaction , thalassemia , multiplex , polymerase chain reaction optimization , chemistry , microbiology and biotechnology , chromatography , computational biology , biology , genetics , biochemistry , gene
Capillary electrophoresis (CE) is the most useful tool for DNA separation because of its high resolution. In this study, different kinds of polymers were used to evaluate the separation efficiency by analyzing a 200‐bp DNA ladder. Under optimized CE conditions, the CE separation was performed by DB‐17 capillary. The running buffer was a 1× TBE buffer containing 0.6% w/v poly(ethylene oxide) (PEO) (Mw: 8 000 000) and 1 μM YO‐PRO‐1; applied voltage was −10 kV (detector at anode side) and the separation temperature was 25°C. Under these optimal conditions, 15 DNA fragments with sizes ranging from 0.2 to 3.0 kb were resolved within 11.5 min and the RSD of migration time were less than 0.55% ( n =3). This method, combined with three‐step multiplex PCR, was applied to detect five α‐thalassemia deletions, including ‐α 3.7 , ‐α 4.2 , ‐ ‐ SEA , ‐ ‐ FIL and ‐ ‐ THAI . A total of 21 patients diagnosed with α‐thalassemia were analyzed using this developed method and all results agreed with those already obtained by gel electrophoresis.