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Matrix conditioning for lengthened capillary DNA sequencing
Author(s) -
Griess Gary A.,
Hardies Stephen C.,
Serwer Philip
Publication year - 2005
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200406127
Subject(s) - capillary electrophoresis , dna , matrix (chemical analysis) , dna sequencing , chemistry , base pair , analytical chemistry (journal) , chromatography , biochemistry
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.