Validation of the InnoXtract™ DNA extraction method for bone, teeth, and rootless hair.

Forensic casework samples often include human hairs, teeth, and bones. Hairs with roots are routinely processed for DNA analysis, while rootless hairs are either not tested or processed using mitochondrial DNA. Bones and teeth are submitted for human remains identifications for missing persons and mass disaster cases. DNA extraction from these low templates and degraded samples is challenging. The new InnoXtract DNA extraction method utilizes magnetic beads that are optimized to bind small DNA fragments, as small as 100 base pairs, to purify high-yield DNA from compromised samples. This validation study evaluates InnoXtract's ability to obtain amplifiable DNA from samples such as rootless hairs and skeletal remains. Studies performed include sensitivity, stability, repeatability, reproducibility, non-probative samples, and comparison to standard organic extractions. Sensitivity studies demonstrate average yield recoveries ranging from 53% to 100% and 73% to 85% for the InnoXtract hair and bone methods, respectively. Studies demonstrate consistent results across a range of sample types, such as insulted and un-insulted bone and teeth, as well as hair shafts from donors of various ages, gender, race, and hair characteristics. The InnoXtract bone method outperformed organic extraction. The method was successfully automated on a MagMAX™ Express-96, with recoveries over 70% relative to the manual version. InnoXtract has the potential as an automated high-throughput, high-yield bone extraction method with 6 h of total extraction time for up to 96 samples. The validation study results demonstrate that the InnoXtract kits produce high-yield and high-quality DNA from compromised bone, teeth, and hair shaft samples.

Development and validation of a novel method "SpermX™" for high throughput differential extraction processing of sexual assault kits (SAKs) for DNA analysis.

The Sperm X method uses a nanotechnology derived polymer membrane that functions as a separation medium to effectively trap sperm cells while enabling efficient flow through of the digested epithelial cell DNA. This specialized membrane enabled development of a method that could significantly increase a forensic laboratory's ability to obtain male sperm fraction DNA profiles. The SpermX device provides a rapid, reproducible procedure that is easy to implement in a single-tube format as well as high-throughput truly automated hands-free workflows. Validation studies, performed using the manual SpermX method, include sensitivity, stability, precision (reproducibility and repeatability), mixtures, and a method comparison to the traditional differential extraction. Sensitivity and method comparison studies demonstrated a wide range of sperm cells, from a high of over 2.78 million cells (9158 ng) to a low of 25 cells (83 pg), can be trapped by the SpermX membrane. Stability studies on various substrates (i.e., carpet, cotton, denim, polyester, and silk) and degraded semen gave the expected male DNA profiles. Data from the same operator and a different operator were consistent with low variance. Mixtures, with ratios ranging from approximately 10:1-18182:1, created to simulate real casework type samples including buccal/semen, vaginal epithelial/semen, and post coital swabs at different time intervals, were tested. A comparison of the SpermX method to the conventional differential extraction method resulted in comparable probative male profile allelic data and associated statistical probabilities. For low level sperm samples, down to 25 sperm cells (83 pg), the SpermX method outperformed the conventional differential extraction with more genotypic information and associated probabilities.

Screening biological stains with qPCR versus lateral flow immunochromatographic test strips: a quantitative comparison using analytical figures of merit.

Biological fluid identification is an important facet of evidence examination in forensic laboratories worldwide. While identifying bodily fluids may provide insight into which downstream DNA methods to employ, these screening techniques consume a vital portion of the available evidence, are usually qualitative, and rely on visual interpretation. In contrast, qPCR yields information regarding the amount and proportion of amplifiable genetic material. In this study, dilution series of either semen or male saliva were prepared in either buffer or female blood. The samples were subjected to both lateral flow immunochromatographic test strips and qPCR analysis. Analytical figures of merit-including sensitivity, minimum distinguishable signal (MDS) and limit of detection (LOD)-were calculated and compared between methods. By applying the theory of the propagation of random errors, LODs were determined to be 0.05 µL of saliva for the RSID™ Saliva cards, 0.03 µL of saliva for Quantifiler(®) Duo, and 0.001 µL of semen for Quantifiler(®) Duo. In conclusion, quantitative PCR was deemed a viable and effective screening method for subsequent DNA profiling due to its stability in different matrices, sensitivity, and low limits of detection.

STR DNA typing: increased sensitivity and efficient sample consumption using reduced PCR reaction volumes.

Improvements in detection limits/sensitivity and lower sample consumption are potential benefits of reducing PCR reaction volumes used in forensic DNA typing of crime scene samples. This premise was studied first with experimental mixtures and a nine-loci megaplex, which demonstrated stochiometric amplification and accurate detection. Next, adjudicated casework samples were subjected to amplification under 15 different template DNA to PCR reaction volume ratios. Reduction of PCR reaction volume and DNA down to 10 microL and 0.500 ng, respectively, produced identical profiles with the same signal intensity and heterozygous allele peak height ratio (HR). Reduction to 5 microL and 0.063 ng yielded HR values that were slightly affected in one to three STR loci. PCR reaction volume reduction can enhance detection and sensitivity while reducing the consumption of irreplaceable crime scene samples.

Precision studies using the ABI prism 3100 genetic analyzer for forensic DNA analysis.

Precision studies using the ABI Prism 3100 Genetic Analyzer have been conducted by performing multiple runs of the AmpFlSTR Identifiler allelic ladder, 9947A kit positive control DNA and the 250-base-pair fragment from the internal size standard (GeneScan LIZ-500). Intra-run and inter-run precision data demonstrates the sizing reproducibility of the 3100 instrument for forensic applications. This precision data is utilized to assist in making allele assignments for outlier and variant alleles (i.e., 'off-ladder' alleles). It also provides a means of addressing systematic variations due to temperature fluctuations, polymer (POP-4) or capillary effects, and associated troubleshooting efforts.