In this article, capillary electrophoresis of DNA fragments and polymerase chain reaction products in sieving liquid polymers is reviewed. The first part of the review is dedicated to the development of N-substituted acrylamides, which in general guarantee a greater resistance to hydrolytic attack. Among the various monomers developed, an outstanding molecule appears to be N-acryloyl aminopropanol (AAP), which combines an extreme resistance to alkaline hydrolysis (500 times greater than plain acrylamide) with a substantially higher hydrophilicity. Poly(AAP) matrices exhibit a unique behavior in DNA separations, especially at high temperatures (60°C), where most other matrices fail. In separation of antisense oligonucleotides, isoelectric buffers allow very high field strengths (up to 800 V/cm) due to their extremely low conductivity, thus permitting separations on the order of a few minutes. Among such buffers, histidine (pH = pI = 7.47) and lysine (pH = pI = 9.74) appear to be the best ones. Detection of DNA point mutations is accomplished with a peculiar nonisocratic capillary zone electrophoresis (CZE), consisting of creating temporal thermal gradients, analogous to temperature-programmed gas chromatography. A unique approach is described, consisting of creating temperature gradients not with external thermostats, but with internal Joule effects produced by voltage programming (which results in temperature ramps). The review ends with some unique applications of CZE: the possibility of precise gene dosage, relying on on-line detection and quantitation of the separated peaks. Examples of such gene dosages are given, such as assessment of Down's syndrome and determination of homozygous and heterozygous states of RhD blood groups.
ASJC Scopus subject areas
- Molecular Biology