High-resolution melt analysis for the detection of skin/sweat via DNA methylation.

The determination of tissue type is important when reconstructing a crime scene as skin cells may indicate innocent contact, whereas other types of cells, such as blood and semen, may indicate foul play. Up to now, there has been no specific DNA methylation-based marker to distinguish skin cell DNA from other body fluids. The goal of this study is to develop a DNA methylation-based assay to detect and identify skin cells collected at forensic crime scenes for use in DNA typing. For this reason, we have utilized a DNA methylation chip array-based genome-wide association study to identify skin-specific DNA methylation markers. DNA obtained from skin along with other body fluids, such as semen, saliva, blood, and vaginal epithelia, were tested using five genes that were identified as sites for potential new epigenetic skin markers. Samples were collected, bisulfite converted, and subjected to real-time polymerase chain reaction (PCR) with high-resolution melt analysis. In our studies, when using WDR11, PON2, and NHSL1 assays with bisulfite-modified PCR, skin/sweat amplicons melted at lower temperatures compared to blood, saliva, semen, and vaginal epithelia. One-way analysis of variance demonstrates that these three skin/sweat markers are significantly different when compared with other body fluids (p < 0.05). These results demonstrate that high-resolution melt analysis is a promising technology to detect and identify skin/sweat DNA from other body fluids.

Development of a microwave-based extraction for forensic biological samples.

In this study, a quick microwave-based treatment was developed as a front end for DNA analysis of forensic samples. The effect of microwave treatment is to cause cell disruption which can improve the release of DNA during direct PCR as well as with extraction methods. Exposure to microwave preprocessing improved the quality of rapid genotyping, particularly when used with low level samples. Optimal results were obtained when samples were microwaved at 300W for 40 s, resulting in improved allele detection. Overall, the addition of this simple preprocessing step improves sensitivity and allele recovery for low level DNA samples when combined with expedited DNA analysis workflows. Its main advantages include speed, low cost, compatibility with downstream DNA methods and application to a wide variety of samples.

Identifying Methylation Patterns in Dental Pulp Aging: Application to Age-at-Death Estimation in Forensic Anthropology.

Age-at-death estimation constitutes one of the key parameters for identification of human remains in forensic investigations. However, for applications in forensic anthropology, many current methods are not sufficiently accurate for adult individuals, leading to chronological age estimates erring by ±10 years. Based on recent trends in aging studies, DNA methylation has great potential as a solution to this problem. However, there are only a few studies that have been published utilizing DNA methylation to determine age from human remains. The aim of the present study was to expand the range of this work by analyzing DNA methylation in dental pulp from adult individuals. Healthy erupted third molars were extracted from individuals aged 22-70. DNA from pulp was isolated and bisulfite converted. Pyrosequencing was the chosen technique to assess DNA methylation. As noted in previous studies, we found that ELOVL2 and FHL2 CpGs played a role in age estimation. In addition, three new markers were evaluated-NPTX2, KLF14, and SCGN. A set of CpGs from these five loci was used in four different multivariate regression models, providing a Mean Absolute Error (MAE) between predicted and chronological age of 1.5-2.13 years. The findings from this research can improve age estimation, increasing the accuracy of identification in forensic anthropology.

The development of miniSTRs as a method for high-speed direct PCR.

There are situations in which it would be very valuable to have a DNA profile within a short time; for example, in mass disasters or airport security. In previous work, we have promoted reduced size STR amplicons for the analysis of degraded DNA. We also noticed that shorter amplicons are more robust during amplification, making them inhibition resistant, and potentially applicable to high-speed direct PCR. Here, we describe a set of miniSTRs capable of rapid direct PCR amplification. The selected markers are a subset of the Combined DNA Index System (CODIS) loci modified to permit high-speed amplification. Using the proposed protocol, the amplification of eight loci plus amelogenin directly from a saliva sample can be completed in 7 min and 38 s using a two-step PCR with 30 cycles of 98°C for 2 s and 62°C for 7 s on a Streck Philisa thermocycler. Selection of DNA polymerase, optimization of the two-step PCR cycling conditions, the primer concentrations, and the dilution of saliva is described. This method shows great potential as a quick screening method to obtain a presumptive DNA profile when time is limited, particularly when combined with high-speed separation and detection methods.

Development of a body fluid identification multiplex via DNA methylation analysis.

The goal of this study is to develop an epigenetic multiplex for body fluid identification based on tissue specific DNA methylation. A series of genetic loci capable of discerning the origin of DNA as coming from saliva, blood, vaginal epithelia, or semen were used for this application. The markers - BCAS4, CG06379435, VE_8, and ZC3H12D - were amplified together and then sequenced via pyrosequencing. Methylation values for cytosine guanine dinucleotide (CpG) sites at each locus were then measured across the four markers. In total, 124 samples were collected, and bisulfite modified to convert unmethylated DNA to uracil. This converted DNA was then amplified via multiplex PCR with reverse primers containing a biotin molecule. Biotinylated PCR products were then analyzed using pyrosequencing to generate a series of pyrograms containing 18 CpG sites. The percent methylation at each CpG site was determined, and then agglomerative hierarchical cluster analysis was used to create a model to indicate sample origin. Further analysis reduced the number of CpG sites required for optimal determination of body fluid type to five. This study demonstrates an efficient multiplexed body fluid identification process utilizing DNA methylation that can be easily implemented in forensic laboratories.

An aptamer-based paper microfluidic device for the colorimetric determination of cocaine.

A method utilizing paper microfluidics coupled with gold nanoparticles and two anticocaine aptamers has been developed to detect seized cocaine samples. The ready-to-use format involves the use of a paper strip that produces a color change resulting from the salt-induced aggregation of gold nanoparticles producing a visible color change indicating the presence of the drug. This format is specific for the detection of cocaine. The visual LOD for the method was 2.5 µg and the camera based LOD was 2.36 µg. The operation of the device is easy and rapid, and does not require extensive training or instrumentation. All of the materials utilized in the device are safe and environmental friendly. This device should prove a useful tool for the screening of forensic samples.

Separation and identification of antidepressants by acrylate-based monolithic column capillary electrochromatography with UV detection.

This paper details a method for separation and identification of six antidepressant compounds using an acrylate-based porous monolithic capillary. Detection takes place using CEC coupled with UV or mass spectrometric detection. The CEC-UV method provides an effective and efficient method for the separation and identification of the analytes. Spiked urine samples were utilized to check method capability, and a liquid-liquid extraction procedure was developed to perform sample extraction. Detection limits ranged from 10 to 30 ng/mL.

The Application of Voltammetric Analysis of Δ(9) -THC for the Reduction of False Positive Results in the Analysis of Suspected Marijuana Plant Matter.

The development of methodologies using inexpensive, fast, and reliable instrumention is desirable in illicit drug analysis. The purpose of this study was based on cyclic voltammetry technique to differentiate the electrochemical behavior of ∆(9) -THC, the psychoactive substance in marijuana, and five different extract plants to yield false positive results after analysis protocol for cannabinoids using thin-layer chromatography and Fast Blue B salt. After applying a deposition potential of -0.5 V in a glassy carbon working electrode, the results indicated an anodic peak current at 0.0 V versus Ag/AgCl after addition of ∆(9) -THC solution in the electrochemical cell, and limits of detection and quantification were 1.0 ng mL(-1) and 3.5 ng mL(-1) , respectively. Other interfering plants showed distinct amperometric responses. This methodology was useful to detect ∆(9) -THC even in the presence of the Fast Blue B salt, which avoided false positive results for all the studied extract plants.

The use of direct analysis in real time (DART) to assess the levels of inhibitors co-extracted with DNA and the associated impact in quantification and amplification.

The measure of quality in DNA sample processing starts with an effective nucleic acid isolation procedure. Most problems with DNA sample typing can be attributed to low quantity DNA and/or to the presence of inhibitors in the sample. Therefore, establishing which isolation method is best at removing potential inhibitors may help overcome some of the problems analysts encounter by providing useful information in the determination of the optimal approach for any given sample. Direct analysis in real time (DART) mass spectrometry was used in this study to investigate the ability of different extraction methods to remove PCR inhibitors. Methods investigated included both liquid/liquid (phenol-chloroform) and solid phase based robotic procedures, (PrepFiler™ and EZ1 chemistries). Following extraction, samples were analyzed by DART in order to determine the level of remaining inhibitors and then quantified and amplified to determine the effect any remaining inhibitor had on the overall results. The data suggests that organic extraction methods result in detrimental amounts of phenol carryover while automated methods may produce carry-over of bile salts and other chemicals that preferentially bind the solid phase matrix. Both of these effects can have a negative impact in downstream sample processing and genotyping by PCR.

The application of alkaline lysis and pressure cycling technology in the differential extraction of DNA from sperm and epithelial cells recovered from cotton swabs.

This study reports the development of a two-step protocol using pressure cycling technology (PCT) and alkaline lysis for differential extraction of DNA from mixtures of sperm and vaginal epithelial cells recovered from cotton swabs. In controlled experiments, in which equal quantities of sperm and female epithelial cells were added to cotton swabs, 5 min of pressure pulsing in the presence of 0.4 M NaOH resulted in 104 ± 6% recovery of female epithelial DNA present on the swab. Following the pressure treatment, exposing the swabs to a second 5-min alkaline treatment at 95 °C without pressure resulted in the selective recovery of 69 ± 6% of the sperm DNA. The recovery of the vaginal epithelia and sperm DNA was optimized by examining the effect of sodium hydroxide concentration, incubation temperature, and time. Following the alkaline lysis steps, the samples were neutralized with 2 M Tris (pH 7.5) and purified with phenol-chloroform-isoamyl alcohol to permit downstream analysis. The total processing time to remove both fractions from the swab was less than 20 min. Short tandem repeat (STR) analysis of these fractions obtained from PCT treatment and alkaline lysis generated clean profiles of female epithelial DNA and male sperm DNA for 1:1 mixtures of female and male cells and predominant male profiles for mixtures up to 5:1 female to male cells. By reducing the time and increasing the recovery of DNA from cotton swabs, this new method presents a novel and potentially useful procedure for forensic differential extractions.

An investigation of PCR inhibition using Plexor(®) -based quantitative PCR and short tandem repeat amplification.

A common problem in forensic DNA typing is PCR inhibition resulting in allele dropout and peak imbalance. In this paper, we have utilized the Plexor(®) real-time PCR quantification kit to evaluate PCR inhibition. This is performed by adding increasing concentrations of various inhibitors and evaluating changes in melt curves and PCR amplification efficiencies. Inhibitors examined included calcium, humic acid, collagen, phenol, tannic acid, hematin, melanin, urea, bile salts, EDTA, and guanidinium thiocyanate. Results were plotted and modeled using mathematical simulations. In general, we found that PCR inhibitors that bind DNA affect melt curves and CT takeoff points while those that affect the Taq polymerase tend to affect the slope of the amplification curve. Mixed mode effects were also visible. Quantitative PCR results were then compared with subsequent STR amplification using the PowerPlex(®) 16 HS System. The overall results demonstrate that real-time PCR can be an effective method to evaluate PCR inhibition and predict its effects on subsequent STR amplifications.

Chiral separation of 12 cathinone analogs by cyclodextrin-assisted capillary electrophoresis with UV and mass spectrometry detection.

In this study, a rapid chiral separation of 12 cathinones analogs has been developed and validated using cyclodextrin-assisted CE with UV and TOF-MS detection. Optimum separation was obtained on a 57.5 cm × 50 µm capillary using a buffer system consisting of 10 mM ß-cyclodextrin (ß-CD) in a 100 mM phosphate buffer for CE-UV, and 0.6% v/v highly sulfated-γ-cyclodextrin (HS-γ-CD) in a 50 mM phosphate buffer for CE-MS. In the CE-MS experiment, a partial filling technique was employed to ensure that a minimum amount of cyclodextrin entered the mass spectrometer. All analytes were separated within 18 min in the CE-UV separation and identified by TOF-MS. Ten compounds were enantiomerically separated using ß-CD in the UV mode and an additional two more were enantiomerically separated using HS-γ-CD in the MS mode. Detection limits down to 1.0 ng/mL were obtained. The method was then applied to examine seized drugs.

The effect of internal control sequence and length on the response to PCR inhibition in real-time PCR quantitation.

PCR inhibitors can originate from a variety of sources and can co-extract with the DNA template, resulting in reduced amplification and/or dropped alleles. Currently real time PCR is used to provide a check for the presence of PCR inhibition by monitoring the quality of amplification of an internal control. In this paper we examine the effect of internal control length and sequences on its sensitivity to PCR inhibition by varying concentrations of commonly encountered PCR inhibitors. Data from both amplification and melt curves were evaluated. The results show that while amplicon sequence has minor effects on amplification efficiency and melt curves, amplicon length has a more dramatic effect, regardless of inhibitor type. Given the increasing variety of STR typing kits and their documented differences in performance with respect to inhibition, the data obtained in this study can be used to assist designers of real time PCR kits to adjust their internal PCR controls (IPC) to permit a more targeted estimation of inhibition.

Separation and detection of smokeless powder additives by ultra performance liquid chromatography with tandem mass spectrometry (UPLC/MS/MS).

A reversed phase gradient ultra performance liquid chromatography tandem mass spectrometry (UPLC/MS/MS) method has been developed for the analysis of smokeless powders. A total of 20 different components were separated by UPLC and detected by MS/MS in multiple reaction monitoring (MRM) mode. These compounds included diphenylamines, centralites, nitrotoluenes, nitroglycerin, and various phthalates. Simultaneous positive and negative electrospray ionization (ESI) was used along with negative atmospheric pressure chemical ionization (APCI) to detect all compounds in a single analysis. Analysis times were under 8 min with a gradient of 10-73% organic at a flow rate of 0.500 mL/min. With this method, ultraviolet and MRM limits of detection ranging from 0.08 to 2.6 ng and 0.4-64 ng injected were achieved. Commercially available smokeless powders were also extracted with methylene chloride and characterized using the developed UPLC/MS/MS method. The procedure permits the determination of compositional differences between different brands as well as lot-to-lot variations.

Separation and identification of smokeless gunpowder additives by capillary electrochromatography.

This paper details a method for the separation and identification of fourteen organic compounds commonly found as constituents in commercial smokeless powders using a hexyl acrylate-based porous monolith. Capillary electrochromatography (CEC) coupled to UV and time of flight-mass spectrometry (TOF-MS) methods were both explored. The CEC-UV method provides an effective and efficient method for the detection of all components in the additive package of the powder. The TOF-MS procedure provides better sensitivity and selectivity allowing an additional confirmation of the presence of the subset of those compounds, which are detectable via positive and/or negative ion electrospray ionization mass spectrometry. Both methods were used for the analysis of smokeless powder components in a mixed standard as well as in the determination of the composition of the additive package of individual powders.

Rapid and specific detection of urea nitrate and ammonium nitrate by electrospray ionization time-of-flight mass spectrometry using infusion with crown ethers.

Urea nitrate (UN) and ammonium nitrate (AN) are fertilizer-based explosives that are commonly used in improvised munitions and can have highly destructive effects. Because they are in the form of salts, their relatively low volatility makes them difficult to detect at trace levels. In addition, these salts readily undergo metathetic reactions in water to form urea, ammonium and nitrate, which are ubiquitous in the environment. Thus, selective methods are needed for their detection. In this study, a procedure was developed to detect UN and AN in non-aqueous environments by positive ion electrospray ionization mass spectrometry through the addition of 18-crown-6. The method is sensitive, with detection limits under 2 µM, and selective. The procedure is capable of differentiating urea from uronium ions (protonated urea) and a mixture of urea and AN did not interfere with the UN signal. The procedure is quite versatile and the addition of crown ethers to the sample matrix does not interfere with the detection of high explosives in the negative ionization mode. Experimental results are presented on the utilization of the method in the detection of UN and AN on various surfaces. Semi-quantitative studies showed that AN and UN can be detected at trace levels following finger transfer and a series of studies were performed to demonstrate the effect of various interferences. The results show the method to be a quick and robust procedure for trace detection.

A comparison of the effects of PCR inhibition in quantitative PCR and forensic STR analysis.

In this paper we compare the effects of three representative PCR inhibitors using quantitative PCR (qPCR) and multiplex STR amplification in order to determine the effect of inhibitor concentration on allele dropout and to develop better ways to interpret forensic DNA data. We have used humic acid, collagen and calcium phosphate at different concentrations to evaluate the profiles of alleles inhibited in these amplifications. These data were correlated with previously obtained results from quantitative PCR including melt curve effects, efficiency changes and cycle threshold (Ct) values. Overall, the data show that there are two competing processes that result from PCR inhibition. The first process is a general loss of larger alleles. This appears to occur with all inhibitors. The second process is more sequence specific and occurs when the inhibitor binds DNA, altering the cycle threshold and the melt curve. This sequence-specific inhibition results in patterns of allele loss that occur in addition to the overall loss of larger alleles. The data demonstrate the applicability of utilizing real-time PCR results to predict the presence of certain types of PCR inhibition in STR analysis.

The development of mini pentameric STR loci for rapid analysis of forensic DNA samples on a microfluidic system.

There is increasing interest in developing methods for portable DNA analysis in mass disasters and criminal identification. Currently most forensic STR DNA analysis is performed by CE; however, these instruments are not portable and require long sample run times. One potential solution is the development of microfluidic systems for DNA typing. Unfortunately, fairly long (ca. 20 cm) separation channels are usually required for the proper resolution of multiplexed STR loci used in human identification. Commercially available systems like the Agilent 2100 Bioanalyzer have a small footprint and utilize chips with shorter channels and reduced resolution. Such portable systems might be valuable for evidence screening in remote locations. However, due to their lower resolution, most standard 4 base STR loci and their inherent 2 base variants will not resolve on such systems. In this paper, we discuss the development of reduced length pentameric (5 base) STR amplicons. Pentameric STRs have fewer variant alleles and are easier to separate due to the wider spacing between alleles. By incorporating novel denaturing sieving polymers in a short microfluidic channel, we demonstrate efficient separations on these chips. Such an approach can serve as a useful tool for rapid microfluidic DNA typing.

Minimizing the number of voltage sources for pinched injection on a microfluidic device.

This work presents a novel injection design for microfluidic systems which is able to perform pinched injections using a single power supply. This geometry also minimizes the injection time compared to standard pinched injections. This new design was developed and investigated by computer simulation and these results were confirmed by experimental studies using the injection of fluorescent dye.