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 |
|---|---|
| Pages (from-to) | 1506-1511 |
| Number of pages | 6 |
| Journal | Electrophoresis |
| Volume | 15 |
| Issue number | 12 |
| Publication status | Published - 1994 |
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ASJC Scopus subject areas
- Clinical Biochemistry
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Detection of point mutations by capillary electrophoresis in liquid polymers in temporal thermal gradients. / Gelfi, C.; Righetti, P. G.; Cremonesi, L.; Ferrari, M.
In: Electrophoresis, Vol. 15, No. 12, 1994, p. 1506-1511.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Detection of point mutations by capillary electrophoresis in liquid polymers in temporal thermal gradients
AU - Gelfi, C.
AU - Righetti, P. G.
AU - Cremonesi, L.
AU - Ferrari, M.
PY - 1994
Y1 - 1994
N2 - 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.
AB - 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.
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M3 - Article
C2 - 7536669
AN - SCOPUS:0028567010
VL - 15
SP - 1506
EP - 1511
JO - Electrophoresis
JF - Electrophoresis
SN - 0173-0835
IS - 12
ER -