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Fast capillary electrophoresis‐laser induced fluorescence analysis of ligase chain reaction products: Human mitochondrial DNA point mutations causing Leber's hereditary optic neuropathy
Author(s) -
Muth Jochen,
Williams P. Mickey,
Williams Stephen J.,
Brown Michael D.,
Wallace Douglas C.,
Karger Barry L.
Publication year - 1996
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.1150171212
Subject(s) - capillary electrophoresis , ethidium bromide , point mutation , microbiology and biotechnology , mitochondrial dna , dna , ligase chain reaction , leber's hereditary optic neuropathy , chemistry , fluorescence , polymerase chain reaction , genomic dna , biology , mutation , gene , biochemistry , physics , multiplex polymerase chain reaction , quantum mechanics
High speed capillary electrophoresis‐laser‐induced fluorescence (CE‐LIF) has been used to separate and detect point mutations using the ligase chain reaction (LCR). The method utilizes short capillary columns (7.5 cm effective length) and fields of 400 V/cm to analyze DNA‐ethidium bromide complexes using an He/Ne laser. The method was first demonstrated with a commercially available kit for LCR based on a lac I gene fragment inserted in a Blusescript® II phagemid. LCR‐CE‐LIF was then applied to detect point mutations in human mitochondrial DNA. resulting in Leber's hereditary optic neuropathy (LHON). Three severe mutations were analyzed in which the original base is substituted by a thymidine base at positions 3460, 11778 and 14459. Appropriate primers were designed with polyT tails for length discrimination of pooled samples. Successful detection of mutated samples was achieved, with appropriate correction for small amounts of nonspecific ligated product. The method is rapid, easy to implement, and automatale.