Detection of point mutations by capillary electrophoresis in liquid polymers in temporal thermal gradients.
A new and fast method is described for detection of point mutations in polymerase-chain reaction (PCR)-amplified DNA, based on capillary electrophoresis in sieving liquid polymers in presence of temporal thermal gradients. The background electrolyte contains a constant amount of denaturing agent (e.g., 6 M urea) and the DNA fragments are injected in a constant-temperature plateau below the melting temperature (Tm). After loading, a temperature ramp is activated (typically from 0.2 to 0.6 degrees C/min, according to the melting profiles of the DNA duplexes under investigation) with resultant branching of homo- and heteroduplexes at different times along the migration path. In the case of individuals heterozygous for a point mutation, the expected four-band pattern is obtained. The temperature gradient is not produced externally, via circulating coolant and a thermostat, but is generated internally by using a dedicated computer program able to calculate the precise inner temperature under given electric conditions. The method is applied to the identification of three point mutations located in exon 17b (R1066H, R 1066C, F1052V) and of two polymorphisms located in exon 14a (V868V, T854T) of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.
Gelfi
C
C
ITBA, CNR, Milano, Italy.
Righetti
P G
PG
Cremonesi
L
L
Ferrari
M
M
D000225:Adenine / Q000378:metabolism; D003550:Cystic Fibrosis / Q000235:genetics; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D003596:Cytosine / Q000378:metabolism; D004247:DNA; D004586:Electrophoresis / Q000379:methods*; D005091:Exons; D006147:Guanine / Q000378:metabolism; D006579:Heterozygote; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics*; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D011108:Polymers; D011110:Polymorphism, Genetic; D013816:Thermodynamics; D013941:Thymine / Q000378:metabolism
C505032:CFTR protein, human; D008565:Membrane Proteins; D011108:Polymers; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D006147:Guanine; D003596:Cytosine; D004247:DNA; D000225:Adenine; D013941:Thymine