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Dual‐color microchip electrophoresis with single‐photon avalanche diodes: Application to mutation detection
Author(s) -
Stenirri Stefania,
Cretich Marina,
Rech Ivan,
Restelli Alessandro,
Ghioni Massimo,
Cova Sergio,
Ferrari Maurizio,
Cremonesi Laura,
Chiari Marcella
Publication year - 2008
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.200800323
Subject(s) - amplicon , microbiology and biotechnology , mutation , biology , mutant , dna sequencer , genetics , allele , genotype , dna , polymerase chain reaction , gene
A novel microchip electrophoresis instrument based on single‐photon avalanche diodes was used for the molecular characterization of mutations in disease genes. The identification of the main mutation causing cystic fibrosis, named ΔF508, by the Amplification Refractory Mutation System was used to validate the technology. In our implemented protocol the wild‐type and mutant allele‐specific primers are labeled with Cy5 and Cy5.5, respectively. The protocol enables the amplification of the DNA sample in a single PCR. The genotype was deduced from the fluorescence of the amplicons run in the CE microchip. Validation on 15 DNA samples from either homozygous wild‐type or heterozygous and homozygous mutated control subjects proved the complete reliability of the system, thus confirming its high diagnostic potential.