Forensic DNA analysis on microfluidic devices: a review.

The advent of microfluidic technology for genetic analysis has begun to impact forensic science. Recent advances in microfluidic separation of short-tandem-repeat (STR) fragments has provided unprecedented potential for improving speed and efficiency of DNA typing. In addition, the analytical processes associated with sample preparation--which include cell sorting, DNA extraction, DNA quantitation, and DNA amplification--can all be integrated with the STR separation in a seamless manner. The current state of these microfluidic methods as well as their advantages and potential shortcomings are detailed. Recent advances in microfluidic device technology, as they pertain to forensic DNA typing, are discussed with a focus on the forensic community.

Development of a human-specific real-time PCR assay for the simultaneous quantitation of total genomic and male DNA.

A duplex real-time quantitative PCR assay was developed for forensic DNA analysis, which provides simultaneous quantitation of total genomic human DNA and human male DNA. The assay utilizes two spectrally resolved fluorogenic probes in a 5' nuclease (TaqMantrade mark) assay. Within the range of organisms empirically tested and based upon theoretical specificity using National Center for Biotechnology Information GenBank sequences, primer and probe sequences were shown to be human specific, and the Y-chromosome probe, male-specific. A mixture-challenge study resulted in accurate quantitation of 25 pg male DNA in a mixture of up to 1:5000 (male:female DNA). Additional experimental results include comparisons with the slot blot method and commercial real-time PCR kits. The assay developed addresses the shortcomings of the traditional slot blot method as well as the commercial real-time PCR kits. This method is shown to be specific, relatively simple, rapid, has low limits of detection, and consumes limited sample in addition to reporting both the male and total genomic DNA concentrations present.

Chemical composition of fingerprints for gender determination.

This work investigates the chemical nature of fingerprints to ascertain whether differences in chemical composition or the existence of chemical markers can be used to determine personal traits, such as age, gender, and personal habits. This type of information could be useful for reducing the pool of potential suspects in criminal investigations when latent fingerprints are unsuitable for comparison by traditional methods. Fingertip residue that has been deposited onto a bead was extracted with a solvent such as chloroform. Samples were analyzed by gas chromatography/mass spectrometry (GC/MS). The chemical components identified include fatty acids, long chain fatty acid esters, cholesterol and squalene. The area ratios of ten selected components relative to squalene were calculated for a small preliminary experiment that showed a slight gender difference for three of these components. However, when the experiment was repeated with a larger, statistically designed experiment no significant differences between genders were detected for any of the component ratios. The multivariate Hotelling's T2 test that tested all ten-component ratios simultaneously also showed no gender differences at the 5% significance level.

Method for determining intracapillary solution temperatures: application to sample zone heating for enhanced fluorescent labeling of proteins.

A fundamental premise in CE relies heavily on the assumption that temperature within the capillary is accurately known and controlled. Theoretical calculations for sample zone and BGE temperature during voltage application are presented. We propose that transient elevation of the sample zone temperature allowed for denaturing and renaturing of proteins in the presence of a fluorescent dynamic labeling reagent. Comparison with the extent of labeling possible with standard on-column dynamic labeling in the absence of elevated temperatures showed order-of-magnitude increases in the fluorescence detection sensitivity of proteins with low surface hydrophobicity. As a result, this represents an example where excess heating in the sample zone during electrophoresis can be exploited advantageously.

Separation of sperm and epithelial cells in a microfabricated device: potential application to forensic analysis of sexual assault evidence.

Forensic DNA analysis of sexual assault evidence requires separation of DNA from epithelial (victim) and sperm (perpetrator) cells. The conventional method used by crime laboratories, which is termed "differential extraction", is a time-consuming process. To supplant the conventional process, separation of sperm from a biological mixture containing epithelial cells has been demonstrated on a microfluidic device. This separation utilizes the differential physical properties of the cells that result in settling of the epithelial cells to the bottom of the inlet reservoir and subsequent adherence to the glass substrate. As a result, low flow rates can be used to separate the sperm cells from the epithelial cell-containing biological mixture. Following cell separation on the microdevice, DNA extraction, amplification, and separation were performed using conventional laboratory methods, showing that the cell separation product in the outlet reservoir was of male origin. The reported cell separation has the potential to impact the forensic DNA analysis backlog of sexual assault cases by circumventing the time-consuming conventional differential extraction procedure.

Hydroxypropyl cellulose as an adsorptive coating sieving matrix for DNA separations: artificial neural network optimization for microchip analysis.

Effective DNA separations in microelectrophoretic systems are complicated by the need to passivate the surface dynamically or covalently. We describe the optimization and utilization of a novel buffer system for fast DNA separations by capillary and microchip electrophoresis without the need for any surface modification or conditioning prior to separation. At concentrations as high as 5%, hydroxypropyl cellulose (HPC) has a relatively low viscosity, allowing for microchip channel filling to be performed with ease. A MES/TRIS buffer system at pH 6.1 eliminates the need for surface preconditioning procedures due to the promotion of hydrogen bonding of HPC with the wall. An additional benefit with this buffer system is the low current observed at high fields when compared to other common DNA separation buffers. An artificial neural network (ANN) was used to model the data and to predict the optimum conditions. Utility of the ANN-optimized system for molecular diagnostic testing was demonstrated by performing microchip separations on DNA samples from patients suspected of having genetic mutations associated with Duchenne muscular dystrophy (DMD). Microchip analysis easily allowed for the patient samples positive for DMD mutations to be distinguished from patient samples negative for the disease.