Integrated Sample Cleanup-Capillary Electrophoresis Microchip for High-Performance Short Tandem Repeat Genetic Analysis

UCSF/UCB Joint Graduate Group in Bioengineering, University of California, Berkeley, California 94720, USA.
Analytical Chemistry (Impact Factor: 5.64). 01/2009; 81(1):210-7. DOI: 10.1021/ac8018685
Source: PubMed

ABSTRACT An integrated PCR sample cleanup and preconcentration process is developed for forensic short tandem repeat (STR) analysis using a streptavidin-modified photopolymerized capture gel injector for microchip capillary electrophoresis (microCE). PCR samples generated with one biotinylated primer and one fluorescent primer provide the input to the streptavidin-based affinity capture-microCE device. Monoplex PCR samples processed by the device exhibited approximately 10- to 50-fold increased fluorescence intensities, and DNA profiles generated using 9-plex STR samples displayed approximately 14- to 19-fold higher signal intensities compared to those analyzed using traditional cross injection. Complete STR profiles were obtained with as few as 25 copies of DNA template using the capture-microCE device. Four DNA samples with various degrees of degradation were also tested. Samples analyzed using the capture-microCE device resulted in a significant increase of successful allele detection. The ability of our capture-microCE device and method to remove contaminating ions, to concentrate the sample injection plug, and to eliminate electrokinetic injection bias provides a powerful approach for integrating sample cleanup with DNA separation.

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    • "These advantages provide a 25-fold increase in separation efficiency yielding efficient multiplexed assays. Oligonucleotide capture regions have also been created using PA gels copolymerized with streptavidin (Yeung et al 2009). Subsequent to PA gel polymerization, biotinylated oligonucleotides were immobilized via streptavidin in the gel regions, thus avoiding the need to copolymerize oligonucleotides directly into the gel matrix. "
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