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

ArticleinAnalytical Chemistry 81(1):210-7 · January 2009with10 Reads
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.
    • "The synthesis and optimization of two linear polyacrylamide matrices for the capillary electrophoresis separation of DNA fragments with less than 70 bases was reported and applied to size PCR markers for wild-type and mutant gastric cancer tissues with a resolution below five bases [35]. A 5 % linear polyacrylamide matrix was used in an integrated microfluidic lab-on-a-chip platform for DNA extraction, amplification, separation, and detection from a crude biological sample, and a full profile of short tandem repeats (STRs) was obtained for a standard DNA template in a 40-min analysis time [34] . Other microfluidic platforms utilizing linear polyacrylamide were employed for the analysis of E. coli363738, Staphylococcus aureus [37, 39], Salmonella typhimurium [37], human respiratory viruses [40], Alu insertions used for gender and ethnicity determination [41, 42], p53 gene mutations [43] , and EndoV/DNA ligase muta- tions [44]. "
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    • "In comparison, the PowerPlex 16 and Profiler Plus chemistries required 34 min or 30 min, respectively for separation and detection on a commercial ABI 310CE as reported from Yeung et al. [88]. Additional work has further shown the capabilities of mCAE devices and has demonstrated rapid ($30 min) separation of high quality genomic and mitochondrial DNA sequencing analysis with 1 bp resolution, and with 99% accuracy of 96 samples simultaneously899091. Yeung et al. in 2009 used a biotin primer modified STR 9plex on a 4 channel mCAE device with a 10 cm effective separation length and determined a limit of detection of 25 copies required to still generate a full STR profile [92]. The mCAE device was additionally reported to successfully genotype 47 single source buccal samples as well as 19 non-probative casework samples for PowerPlex 16 and Power- Plex Y STR chemistries [93]. "
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