Detection of point mutations by capillary electrophoresis in liquid polymers in temporal thermal gradients

C. Gelfi, P. G. Righetti, L. Cremonesi, M. Ferrari

Research output: Contribution to journalArticle

70 Citations (Scopus)

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 languageEnglish
Pages (from-to)1506-1511
Number of pages6
JournalElectrophoresis
Volume15
Issue number12
Publication statusPublished - 1994

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Capillary electrophoresis
Capillary Electrophoresis
Point Mutation
Thermal gradients
Polymers
Hot Temperature
Temperature
Exons
DNA
Liquids
Thermostats
Cystic Fibrosis Transmembrane Conductance Regulator
Polymerase chain reaction
Polymorphism
Coolants
Electrolytes
Melting point
Urea
Computer program listings
Nucleic Acid Denaturation

ASJC Scopus subject areas

  • Clinical Biochemistry

Cite this

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 journalArticle

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