Detection of mutations by fluorescence-assisted mismatch analysis (FAMA).
Fluorescence-assisted mismatch analysis (FAMA) methodology uses bifluorescent double-stranded DNA substrates to maximize the reliability and sensitivity of mutation-scanning procedures that rely on cleavage of mismatches using chemical. This unit presents a nested PCR procedure to fluorescently label each DNA strand, followed by chemical cleavage to detect the point mutations. Fluorescent end labeling with strand-specific fluorophores, electrophoretic separation of cleavage products in an automated Perkin-Elmer ABI 373 or 377 DNA sequencer and analysis using the Perkin-Elmer GENESCAN software expands sensitivity by highlighting weak signals through superimposition of strand-specific fluorescence electropherograms for different samples.
Tosi
M
M
Institut Pasteur, Paris, France.
Verpy
E
E
Meo
T
T
D020137:Base Pair Mismatch; D004252:DNA Mutational Analysis / Q000379:methods*; D005456:Fluorescent Dyes; D005826:Genetics, Medical; D006801:Humans; D009692:Nucleic Acid Heteroduplexes / Q000235:genetics / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA
D005456:Fluorescent Dyes; D009692:Nucleic Acid Heteroduplexes