Fast capillary electrophoresis-laser induced fluorescence analysis of ligase chain reaction products: human mitochondrial DNA point mutations causing Leber's hereditary optic neuropathy.
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 lacI gene fragment inserted in a Bluescript 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 automatable.
Muth
J
J
Barnett Institute, Northeastern University, Boston, MA 02115, USA.
Williams
P M
PM
Williams
S J
SJ
Brown
M D
MD
Wallace
D C
DC
Karger
B L
BL
D001483:Base Sequence; D011088:DNA Ligases / Q000378:metabolism*; D004272:DNA, Mitochondrial / Q000032:analysis* / Q000737:chemistry; D019075:Electrophoresis, Capillary / Q000379:methods*; D005453:Fluorescence; D006801:Humans; D007834:Lasers; D015418:Optic Atrophies, Hereditary / Q000235:genetics*; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D013698:Templates, Genetic
D004272:DNA, Mitochondrial; D011088:DNA Ligases