Capillary electrophoresis with laser-induced fluorescence detection for fast and reliable apolipoprotein E genotyping.
The use of capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection for the rapid determination of apolipoprotein E (apoE) genotypes was studied. High resolution and sensitive detection of the concerned DNA restriction fragments was achieved using CE buffers with hydroxypropylmethylcellulose (HPMC) as sieving polymer and ethidium bromide (EB) as fluorescent intercalating agent. In order to achieve adequate resolutions in short analysis times, parameters such as concentration of HPMC and EB, separation voltage, and length and coating of the capillary were evaluated. Using a separation buffer with 0.8% (w/w) HPMC and 7 microM EB, characteristic DNA-fragment profiles could be obtained for all common apoE genotypes at an overall rate of ten samples per hour. The method allows direct injection of untreated PCR samples and the use of standard fused-silica capillaries which are effectively coated following a short, one-step rinse procedure. With a simple computerized algorithm based on migration-time ratios for pattern assignment, highly reliable apoE genotyping was achieved. Overall, in terms of speed, ease of use and objectivity the presented method provides a significant improvement over previously reported CE-based procedures for apoE genotyping.
Somsen
G W
GW
Department of Pharmaceutical Analysis, University of Groningen, P.O. Box 196, 9700 AD Groningen, The Netherlands. g.w.somsen@pharm.uu.nl
Welten
H T M E
HT
Mulder
F Ph
FP
Swart
C W
CW
Kema
I P
IP
de Jong
G J
GJ
D001057:Apolipoproteins E / Q000235:genetics*; D001483:Base Sequence; D017931:DNA Primers; D019075:Electrophoresis, Capillary / Q000379:methods*; D005838:Genotype; D007834:Lasers; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence / Q000379:methods*
D001057:Apolipoproteins E; D017931:DNA Primers