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
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]. "
    [Show abstract] [Hide abstract] ABSTRACT: This review of capillary electrophoresis methods for DNA analyses covers critical advances from 2009 to 2014, referencing 184 citations. Separation mechanisms based on free-zone capillary electrophoresis, Ogston sieving, and reptation are described. Two prevalent gel matrices for gel-facilitated sieving, which are linear polyacrylamide and polydimethylacrylamide, are compared in terms of performance, cost, viscosity, and passivation of electroosmotic flow. The role of capillary electrophoresis in the discovery, design, and characterization of DNA aptamers for molecular recognition is discussed. Expanding and emerging techniques in the field are also highlighted.
    Full-text · Article · May 2015
    • "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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    Full-text · Article · Apr 2015
    • "Mars, Europa, Enceladus) whether life ever evolved there or not, the ultralow limits of detection provided by this method may be crucial in achieving meaningful results. Additionally, CE is well-suited for integration with other microfabricated components including heaters [8,9], resistive temperature detectors [8,9], pH sensitive electrodes [10], membrane microvalves111213 , etc., on a single device. The integration of networks of monolithic membrane microvalves, in particular, has enabled the automation required for spaceflight-ready liquid-based organic chemical analyses [1,14]. "
    [Show abstract] [Hide abstract] ABSTRACT: Networks of monolithic membrane microvalves integrated into microdevices enable complete automation of liquid-based chemical analyses necessary for fully automated applications, such as spaceflight. Although individual pumping devices and operational routines have been characterized, to date there has been no rigorous evaluation of microvalve layout and its effect on fluidic transfer. Here, we evaluate two microdevices at opposite extremes of fluidic resistance and evaluate three pumping routines on each device. Delay times between operational steps are optimized for fastest fluidic transfer. A 3-valve double-chamber routine enables fastest pumping rates on both devices. On low fluidic resistance devices, a 2-valve (bivalve) pumping routine enables faster fluidic transfer than a 3-valve single-chamber pumping routine. Additionally, low fluidic resistance devices enable significantly faster fluidic transfer (4–6 fold) than their higher resistance counterparts. Back-contamination is qualitatively characterized for the optimized routines; higher fluidic resistance between the pumping architecture and the fluidic output reservoir is the most essential feature for preventing back-contamination. We use these results to suggest design rules to guide future pumping architectures to enable the rapid, contamination-free fluidic transfer that will be necessary in spaceflight applications.
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