Capillary electrophoresis for the detection of known point mutations by single-nucleotide primer extension and laser-induced fluorescence detection.
Capillary electrophoresis (CE) with laser-induced fluorescence (LIF) was used to detect known point mutations using the method of single-nucleotide primer extension (SNuPE). Three different point mutations in human mitochondrial DNA associated with Leber's hereditary optic neuropathy (LHON) were detected by annealing a primer immediately 5' to the mutation on the template and extending the primer by one fluorescently labeled dideoxy terminator complementary to the mutation. By using two or more differently labeled terminators, both the mutant and wild type could be simultaneously detected. The advantages of using CE-LIF for detecting SNuPE reactions include speed and ease of analysis, absence of radioactivity, and potential for automation.
Piggee
C A
CA
Barnett Institute, Northeastern University, Boston, MA 02115, USA.
Muth
J
J
Carrilho
E
E
Karger
B L
BL
D001483:Base Sequence; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers / Q000737:chemistry; D004272:DNA, Mitochondrial / Q000032:analysis* / Q000235:genetics; D019075:Electrophoresis, Capillary / Q000379:methods*; D007834:Lasers; D017354:Point Mutation / Q000235:genetics*; D013050:Spectrometry, Fluorescence; D013698:Templates, Genetic
D017931:DNA Primers; D004272:DNA, Mitochondrial