High-resolution chromatography of nucleic acids on the gen-pak fax column

Deborah J. Stowers, Jenny M B Keim, Prem S Paul, Young S. Lyoo, Michael Merion, Robert M. Benbow

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

High-performance liquid chromatography (HPLC) on a Gen-Pak FAX column has been used to separate and purify microgram amounts of single- and double-stranded DNA and RNA molecules. HPLC of mixtures of DNA restriction fragments showed that fragments within the size range 0.125-23.1 kilobase were easily resolved. Supervoiled (form I) plasmid DNA molecules were readily separated from single-stranded circular DNA of the same length and from various DNA conformational isomers including nicked (form II) and linear (form III) species. Topological isomers generated from supercoiled plasmid DNA molecules by DNA topoisomerase I exhibited different retention times than supercoiled molecules. Supercoiled (form I) DNA molecules were resolved from fully relaxed (form IV) molecules. Synthetic oligonucleotides of 74 and 128 nucleotides in length were separated from failure sequences, as well as from other contaminating synthesis products. Single-stranded circular M13mp18 DNA molecules sufficiently pure for use in automated DNA sequencing systems were prepared by HPLC on a Gen-Pak FAX column. HPLC was also used to fractionate linear double-stranded porcine rotavirus genomic RNA fragments into size classes between 0.3 and 3 kilobase. Finally, HPLC of unfractionated Escherichia coli tRNA molecules resolved multiple species. In all cases, HPLC on Gen-Pak FAX was carried out in phosphate or Tris buffers at neutral pH in the presence of sodium chloride. Columns were not damaged by repeated exposure to impure samples, provided they were cleaned frequently with sodium hydroxide and acetic acid. Although procedures for resolution of the various size ranges for each class of DNA and RNA molecules require further optimization, our preliminary data on the separations obtained, the moderate salt concentrations employed, and the durability of the matrix suggest that this column merits further study.

Original languageEnglish (US)
Pages (from-to)47-65
Number of pages19
JournalJournal of Chromatography A
Volume444
Issue numberC
DOIs
StatePublished - Jul 1 1988

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Chromatography
Nucleic Acids
High Pressure Liquid Chromatography
High performance liquid chromatography
Molecules
Single-Stranded DNA
DNA
Circular DNA
Plasmids
RNA
Superhelical DNA
Isomers
Sodium Hydroxide
Type I DNA Topoisomerase
Tromethamine
Double-Stranded RNA
Rotavirus
Transfer RNA

ASJC Scopus subject areas

  • Analytical Chemistry
  • Clinical Biochemistry
  • Molecular Medicine

Cite this

Stowers, D. J., Keim, J. M. B., Paul, P. S., Lyoo, Y. S., Merion, M., & Benbow, R. M. (1988). High-resolution chromatography of nucleic acids on the gen-pak fax column. Journal of Chromatography A, 444(C), 47-65. https://doi.org/10.1016/S0021-9673(01)94008-7

High-resolution chromatography of nucleic acids on the gen-pak fax column. / Stowers, Deborah J.; Keim, Jenny M B; Paul, Prem S; Lyoo, Young S.; Merion, Michael; Benbow, Robert M.

In: Journal of Chromatography A, Vol. 444, No. C, 01.07.1988, p. 47-65.

Research output: Contribution to journalArticle

Stowers, DJ, Keim, JMB, Paul, PS, Lyoo, YS, Merion, M & Benbow, RM 1988, 'High-resolution chromatography of nucleic acids on the gen-pak fax column', Journal of Chromatography A, vol. 444, no. C, pp. 47-65. https://doi.org/10.1016/S0021-9673(01)94008-7
Stowers, Deborah J. ; Keim, Jenny M B ; Paul, Prem S ; Lyoo, Young S. ; Merion, Michael ; Benbow, Robert M. / High-resolution chromatography of nucleic acids on the gen-pak fax column. In: Journal of Chromatography A. 1988 ; Vol. 444, No. C. pp. 47-65.
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