Premium
Capillary Electrophoresis as a Method to Study DNA Reassociation
Author(s) -
Li Yuanqian,
White Jennifer,
Stokes David,
Sayler Gary,
Sepaniak Michael
Publication year - 2001
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp010005f
Subject(s) - dna , ethidium bromide , capillary electrophoresis , electrophoresis , chemistry , gel electrophoresis , fluorescence , chromatography , base pair , microbiology and biotechnology , biology , biochemistry , physics , quantum mechanics
To develop analytical methodology to assess the genetic complexity of a DNA sample, capillary electrophoresis with laser‐induced fluorescence detection is used to monitor the annealing process of DNA samples. Coated columns are filled with an entangled polymer solution shown to optimally separate DNA through size‐selective capillary electrophoresis. DNA samples are denatured by heating in a boiling water bath for ∼10 min and then cooled to ∼25 °C below the melting point of the DNA sample to initiate the reassociation process. The DNA is detected by means of the laser‐induced fluorescence of intercalated ethidium bromide, which produces a substantially greater signal for double‐ versus single‐stranded DNA. The rate of reassociation is dependent upon the rate at which complimentary strands of DNA encounter each other and the degree of repeating base sequences in the sample (hence, the diversity of the DNA). Experimental parameters also influence the reassociation rate. The effects of salt concentration and incubation temperature are presented. Traditional plots of C o t ( C o = DNA concentration and t = reassociation time) versus % recovery of double‐stranded DNA signal are generated for ΦX 174 Hae III digest and 50 bp stepladder DNA, individually and combined, to calculate the reassociation rate constants for these samples. Because reassociation of individual fragments is observed by the CE‐LIF method, more information about the samples is available than with less specific and time‐consuming traditional methods of investigating DNA reassociation.