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Detection of point mutations by capillary electrophoresis in liquid polymers in temporal thermal gradients
Author(s) -
Gelfi Cecilia,
Righetti Pier Giorgio,
Cremonesi Laura,
Ferrari Maurizio
Publication year - 1994
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.11501501215
Subject(s) - capillary electrophoresis , capillary action , polymer , chromatography , electrophoresis , thermal , chemistry , analytical chemistry (journal) , materials science , physics , thermodynamics , organic chemistry , composite material
A new and fast method is described for detection of point mutations in polmerase‐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 ( T m ). After loading, a temperature ramp is activated (typically from 0.2 to 0.6°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 exo 17b (R1066H, R 1066C, F1052V) and of two polyrmorphisms located in exon 14a (V868V, T854T) of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.