Analysis of two multiplexed short tandem repeat systems using capillary electrophoresis with multiwavelength fluorescence detection.
A series of experiments was performed to analyze the utility of capillary electrophoresis (CE) with multiwavelength detection capabilities for multiplex typing of short tandem repeat loci. Characteristics of the sieving polymer, hydroxyethylcellulose, which affect resolution of single strand (ss) DNA fragments were examined. Additionally, the effects of denaturant in the polymer system, separation voltage, and analysis temperature were studied to ascertain their effects on DNA separations and capillary lifetime. The use of elevated run temperature (60 degrees C) was found to improve sizing precision, to increase the lifetime of capillaries (100 runs or more per capillary), and to provide runtimes of under 20 min. Finally, 100 individual human DNA samples were typed successfully using CE. The average resolution obtained was 1.4 bases for a 200 base fragment with a standard deviation of sizing of 0.2 bases, allowing all alleles examined to be distinguished clearly.
Isenberg
A R
AR
Department of Chemistry, University of Virginia, Charlottesville, USA.
Allen
R O
RO
Keys
K M
KM
Smerick
J B
JB
Budowle
B
B
McCord
B R
BR
D004277:DNA, Single-Stranded / Q000737:chemistry / Q000302:isolation & purification; D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D009691:Nucleic Acid Denaturation; D012091:Repetitive Sequences, Nucleic Acid; D013050:Spectrometry, Fluorescence
D004277:DNA, Single-Stranded