Analysis of two multiplexed short tandem repeat systems using capillary electrophoresis with multiwavelength fluorescence detection

Abstract

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.

Capillary electrophoresis with dual laser detection in separation of amplified fragment length polymorphism fragments

“…The size of each allele is determined by comparing its migration time to a regression line calculated from the size/migration relationship of the several or more than ten internal standard peaks. Using this system for sizing yields an SD of the estimated allele size of 0.2 bases (0.2% RSD), or better [19,20]. Based on our results we obtained RSD a 0.5%.…”

Forensic DNA typing by capillary electrophoresis using the ABI Prism 310 and 3100 genetic analyzers for STR analysis

“…The theory of gel-based separations in CE generally ignores the capillary wall as a contributor to the separation, but under certain conditions the wall can play a major role in the quality of the separation [45]. One effect, which could lead to this type of behavior, is EOF.…”

Capillary electrophoresis at the omics level: Towards systems biology

“…CE has also been developed as a tool for high-throughput analysis. Multiplexed CE systems and multichannel microfluidic devices which use an UV or LIF detector have been designed for high-throughput research [117][118][119][120][121]. Yeung and co-workers [117] applied multiplexed CE to b-lactoglobulin peptide mapping using two different channels in a 96-capillary array.…”