PMID: 7536669Dec 1, 1994Paper

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

Electrophoresis
C GelfiM Ferrari

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 (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 conduc...Continue Reading

References

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Citations

Feb 12, 1998·Electrophoresis·P G Righetti, C Gelfi
Feb 10, 1998·Electrophoresis·F von Heeren, W Thormann
Dec 30, 2004·Electrophoresis·Piotr Kozlowski, Wlodzimierz J Krzyzosiak
Dec 22, 1995·Journal of Chromatography. a·C GelfiP G Righetti
Jan 26, 1996·Journal of Chromatography. B, Biomedical Applications·A W KuypersE J Mensink
Nov 22, 1997·Trends in Biotechnology·K R MitchelsonL J Kricka
Jun 29, 2000·Journal of Chromatography. B, Biomedical Sciences and Applications·J Ren
Dec 10, 1997·Journal of Chromatography. B, Biomedical Sciences and Applications·P G Righetti, C Gelfi
Oct 29, 1997·Journal of Chromatography. B, Biomedical Sciences and Applications·P G Righetti, C Gelfi
Oct 29, 1997·Journal of Chromatography. B, Biomedical Sciences and Applications·R LehmannH M Liebich
Jun 17, 1998·Forensic Science International : Synergy·P G Righetti, C Gelfi
Jul 23, 2004·The Journal of Molecular Diagnostics : JMD·Cecily P Vaughn, Kojo S J Elenitoba-Johnson
Oct 30, 2010·Nature Protocols·Khalid K AlharbiIan N M Day
Jun 4, 2005·Electrophoresis·Jens Bjørheim, Per Olaf Ekstrøm
Apr 12, 2007·Electrophoresis·Maike SniehottaHarald Mischak
Jan 31, 2009·FEMS Microbiology Reviews·Janmejay PandeyRakesh K Jain
Jan 19, 2011·Laboratory Investigation; a Journal of Technical Methods and Pathology·Huidan ZhangJin Fang
Nov 25, 2003·Expert Review of Molecular Diagnostics·Kathleen M Murphy, Karin D Berg
Nov 2, 2007·Chemical Reviews·Karel Kleparník, Petr Bocek

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