Abstract
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 (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-hand 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.
Original language | English |
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Pages (from-to) | 1506-1511 |
Number of pages | 6 |
Journal | Electrophoresis |
Volume | 15 |
Issue number | 12 |
Publication status | Published - 1994 |
ASJC Scopus subject areas
- Clinical Biochemistry