Recent advances in capillary zone electrophoresis of DNA

Pier Giorgio Righetti, Cecilia Gelfi

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

The present minireview summarizes recent developments in the field of DNA separations by capillary zone electrophoresis (CZE), as developed by our group. Separation of antisense oligonucleotides in sieving liquid polymers in isoelectric buffers is discussed first. It is shown that the use of isoelectric buffers, permits very high voltage gradients (up to 1000 V cm-1) with much reduced transit times and increased resolution of all truncated and failed sequences. Oligonucleotides can also be analysed by zone electrophoresis against a stationary pH gradient (typically a pH 6.5-10 range): if injected at the alkaline end, the sample components experience stacking and zone sharpening due to modulation of charge as the oligonucleotides move along the pH gradient. Oligonucleotides having the same length, but differing by one single nucleotide in the chain, can be separated in free solution (i.e. in the absence of a sieving matrix) at strongly acidic pH values (pH 3.0-3.3) where charge differences due to base protonation are maximized. By working in free solution, it has also been possible to measure accurately the free mobility of DNAs, shown to reach a constant value of 3.75 ± 0.04.10-4 cm2 V-1 s-1 at 25°C and in Tris-acetate-EDTA buffer, pH 8.3, above a critical length of ca. 400 base pairs. Finally, detection of point mutations in human genomic DNA is proven to be feasible in nonisocratic CZE, by running temperature-programmed CZE. The temperature gradient is activated within the capillary lumen by voltage ramps during the run, by exploiting joule effects. This technique has been proven to work for all point mutants, from low-, to intermediate-, to high-melters and has been applied to a number of point mutants in cystic fibrosis and thalassemia.

Original languageEnglish
Pages (from-to)239-250
Number of pages12
JournalForensic Science International
Volume92
Issue number2-3
DOIs
Publication statusPublished - Apr 5 1998

Fingerprint

Capillary Electrophoresis
Electrophoresis
Oligonucleotides
Proton-Motive Force
DNA
Buffers
Architectural Accessibility
Temperature
Thalassemia
Antisense Oligonucleotides
Protonation
Electric potential
Point Mutation
Cystic Fibrosis
Base Pairing
Running
Thermal gradients
Polymers
Nucleotides
Modulation

Keywords

  • Capillary electrophoresis
  • DNA mobility
  • DNA point mutations
  • Isoelectric buffers
  • Oligonucleotides

ASJC Scopus subject areas

  • Genetics
  • Analytical Chemistry
  • Pathology and Forensic Medicine
  • Psychiatry and Mental health

Cite this

Recent advances in capillary zone electrophoresis of DNA. / Righetti, Pier Giorgio; Gelfi, Cecilia.

In: Forensic Science International, Vol. 92, No. 2-3, 05.04.1998, p. 239-250.

Research output: Contribution to journalArticle

Righetti, Pier Giorgio ; Gelfi, Cecilia. / Recent advances in capillary zone electrophoresis of DNA. In: Forensic Science International. 1998 ; Vol. 92, No. 2-3. pp. 239-250.
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