Matrix conditioning for lengthened capillary DNA sequencing

Gary A. Griess, Stephen C. Hardies, Philip Serwer

Research output: Contribution to journalArticle

6 Scopus citations

Abstract

Capillary electrophoresis (CE) is currently the preferred format for both DNA sequencing and small DNA fragment analysis. The present study provides a simple revision of the procedure used for CE of DNA with a commercial DNA sequencing apparatus from Applied Biosystems. The revision is electrophoretic conditioning of the sieving matrix (typically POP-6) before sample injection. The effects of this preconditioning are revealed during subsequent analyses performed without replenishing the sieving matrix. The primary effect of preconditioning is to increase peak separations during a subsequent CE. The preconditioning has the following characteristics: (i) The effect on peak separation progressively increases as the preconditioning time increases to at least 6 h. (ii) The effect on peak separation scales approximately as the product of the preconditioning time and the magnitude of the electrical field (162 - 320 V/cm) during preconditioning. (iii) The preconditioning persists for more than 72 h at zero field. Preconditioning of the matrix substantially improves, resolution of fragment analysis in the range of 700-2000 nucleotides. For DNA sequencing, the primary impact of preconditioning is, thus far, extension of the range of low-quality base calls at the end of sequence reading. Matrix preconditioning is a new factor to consider when interpreting data obtained by CE in polymer solutions. The mechanism of preconditioning is not yet known.

Original languageEnglish (US)
Pages (from-to)102-111
Number of pages10
JournalELECTROPHORESIS
Volume26
Issue number1
DOIs
StatePublished - Jan 1 2005

Keywords

  • Capillary electrophoresis
  • DNA sequencing
  • Genomics
  • Hysteresis
  • Polymer solution
  • Sieving

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Clinical Biochemistry

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