Comparison of behavior of N-substituted acrylamides and celluloses on double-stranded DNA separations by capillary electrophoresis at 25° and 60°C

Cecilia Gelfi, Marilena Perego, Fabio Libbra, Pier Giorgio Righetti

Research output: Contribution to journalArticlepeer-review

Abstract

The behavior of N-substituted poly(acrylamides) and of cellulose in the separation of double-stranded DNA by capillary zone electrophoresis (CZE) is evaluated. Contrary to previously held belief, which attributed best separations in poly(acrylamides) to small DNA fragments (typically in the 50-1000 bp size range) and in celluloses to large DNA fragments, we show that also celluloses can achieve fine sieving of short DNA sizes provided they are used at much higher concentrations than previously reported. For example, in the case of hydroxyethylcellulose (HEC), typically used at 0.2-0.8% concentrations, levels of 3% produce excellent patterns, at 25°C, in the 50-600 bp size range. If separations are conducted at 60°C, sieving is lost in most liquid polymers. However, if the concentration of HEC is raised to 6%, and that of hydroxypropylmethylcellulose (HPMC) above 1%, sieving is fully restored. Also, N-substituted acrylamido derivatives, notably N-acryloylaminoethoxyethanol (AAEE) and N-acryloylaminopropanol (AAP) offer excellent performance at both 25° and 60°C, whereas plain poly(acrylamide) irreversibly collapses at high temperatures, possibly due to intrinsic instability of the amido bond under such harsh conditions. Among all the polymers investigated, an 8% solution of poly(AAP) offers the best performance and highest theoretical plates in the 25°-60°C interval. Separations at high temperatures are necessary when dealing with detection of point mutations in temperature-programmed CZE and are preferred in DNA sequencing. Two additional advantages are evident when running DNA separations at 60°C: a marked reduction of analysis times and a linearization of the transit times of the larger (434-587 bp) DNA fragments, in all polymers tested.

Original languageEnglish
Pages (from-to)1342-1347
Number of pages6
JournalElectrophoresis
Volume17
Issue number8
Publication statusPublished - Aug 1996

Keywords

  • Capillary electrophoresis
  • DNA electrophoresis
  • N-Substituted acrylamides

ASJC Scopus subject areas

  • Clinical Biochemistry

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