Individual Identification of Racehorses from Urine Samples Using a 26‐Plex Single‐Nucleotide Polymorphism Assay

To construct a system for identifying individual horses from urine samples that are submitted for postracing doping tests, we developed a genotyping assay based on 26‐plex single‐nucleotide polymorphisms (SNPs). DNA was isolated from urine using a commercially available DNA/RNA extraction kit, and SNP genotyping was achieved with a SNaPshot^? technique. DNA profiles including 26 SNPs were acquired from urine samples and blood/hair samples. Within the studied Thoroughbred population, the 26‐plex assay showed a probability of identity of 5.80 × 10^?11. Compared to the conventional short tandem repeat assay, the SNP assay used less DNA, and the rate of successful genotyping was improved to 97% using aliquots of horse urine as small as 140 μL. The urinary DNA could be successfully genotyped under proper storage concerning refrigeration or freeze–thawing. This SNP assay can be used for individual identification when suspicious results are obtained from horse doping tests.     

Development of a Tetraplex PCR Assay for CYP2D6 Genotyping in Degraded DNA Samples

CYP2D6 polymorphism analysis is gaining increasing interest in forensic pharmacogenetics. Nevertheless, DNA recovered from forensic samples could be of poor quality and not suitable for long polymerase chain reaction required to type CYP2D6 gene prior to SNaPshot minisequencing analysis performed to define alleles with different enzymatic activity. We developed and validated following the guidelines of the Scientific Working Group on DNA Analysis Methods a tetraplex PCR yielding four amplicons of 597, 803, 1142, and 1659 bp encompassing the entire CYP2D6 gene to analyze eleven SNP positions by SNaPshot minisequencing. Concordance, sensitivity, and specificity were assessed. The method, applied to thirty‐two forensic samples failed to amplify with long PCR, allowed the amplification of CYP2D6 gene in 62.5% of degraded samples. The new tetraplex PCR appears a suitable method for CYP2D6 analysis in forensic pharmacogenetics.     

Effectiveness of Single‐Nucleotide Polymorphisms to Investigate Cattle Rustling

Short tandem repeats (STR)s have been the eligible markers for forensic animal genetics, despite single‐nucleotide polymorphisms (SNP)s became acceptable. The technology, the type, and amount of markers could limit the investigation in degraded forensic samples. The performance of a 32‐SNP panel genotyped through OpenArrays^TM (real‐time PCR based) was evaluated to resolve cattle‐specific forensic cases. DNA from different biological sources was used, including samples from an alleged instance of cattle rustling. SNPs and STRs performance and repeatability were compared. SNP call rate was variable among sample type (average = 80.18%), while forensic samples showed the lowest value (70.94%). The repeatability obtained (98.7%) supports the used technology. SNPs had better call rates than STRs in 12 of 20 casework samples, while forensic index values were similar for both panels. In conclusion, the 32‐SNPs used are as informative as the standard bovine STR battery and hence are suitable to resolve cattle rustling investigations.     

A 21‐Locus Autosomal SNP Multiplex and its Application in Forensic Science

To develop a cost‐effective technique for single‐nucleotide polymorphism (SNP) genotyping and improve the efficiency to analyze degraded DNA, we have established a novel multiplex system including 21‐locus autosomal SNPs and amelogenin locus, which was based on allele‐specific amplification (ASA) and universal reporter primers (URP). The target amplicons for each of the 21 SNPs arranged from 63 base pair (bp) to 192 bp. The system was tested in 539 samples from three ethnic groups (Han, Mongolian, and Zhuang population) in China, and the total power of discrimination (TPD) and cumulative probability of exclusion (CPE) were more than 0.99999999 and 0.98, respectively. The system was further validated with forensic samples and full profiles could be achieved from degraded DNA and 63 case‐type samples. In summary, the multiplex system offers an effective technique for individual identification of forensic samples and is much more efficient in the analysis of degraded DNA compared with standard STR typing.     

Development of a Protein‐based Human Identification Capability from a Single Hair

Shed human hair (lacking root nuclear DNA) frequently contributes important information to forensic investigations involving human identification. Detection of genetic variation observed in amino acid sequences of hair proteins provides a new suite of identity markers that augment microscopic hair analysis and mitochondrial DNA sequencing. In this study, a new method that completely dissolves single hairs using a combination of heat, ultrasonication, and surfactants was developed. Dissolved proteins were digested and genetically variant peptide (GVP) profiles were obtained for single hairs (25 mm) via high‐resolution nanoflow liquid chromatography‐based mass spectrometry and a novel exome‐driven bioinformatic approach. Overall, 6519 unique peptides were identified and a total of 57 GVPs were confirmed. Random match probabilities ranged between 2.6 × 10^?2 and 6.0 × 10^?9. The new bioinformatic strategy and ability to analyze GVPs in forensically relevant samples sizes demonstrate applicability of this approach to distinguish individuals in forensic contexts.     

Preliminary Validation of Handheld X‐Ray Fluorescence Spectrometry: Distinguishing Osseous and Dental Tissue from Nonbone Material of Similar Chemical Composition

One of the tasks of a forensic anthropologist is to sort human bone fragments from other materials, which can be difficult when dealing with highly fragmented and taphonomically modified material. The purpose of this research is to develop a method using handheld X‐ray fluorescence (HHXRF) spectrometry to distinguish human and nonhuman bone/teeth from nonbone materials of similar chemical composition using multivariate statistical analyses. The sample materials were derived primarily from previous studies: human bone and teeth, nonhuman bone, nonbiological materials, nonbone biological materials, and taphonomically modified materials. The testing included two phases, testing both the reliability of the instrument and the accuracy of the technique. The results indicate that osseous and dental tissue can be distinguished from nonbone material of similar chemical composition with a high degree of accuracy (94%). While it was not possible to discriminate rock apatite and synthetic hydroxyapatite from bone/teeth, this technique successfully discriminated ivory and octocoral.     

A 27‐Locus STR Assay to Meet All United States and European Law Enforcement Agency Standards,

Different national and international agencies have selected specific STR sets for forensic database use. To enhance database comparison across national and international borders, a 27‐locus multiplex system was developed comprising all 15 STR loci of the European standard set, the current 13 STR loci of the CODIS core, the proposed 22 STR loci of the expanded CODIS core, 4 additional commonly used STR loci, and the amelogenin locus. Development required iterative primer design to resolve primer‐related artifacts, amplicon sizing, and locus‐to‐locus balance issues. The 19.5‐min assay incorporated newly developed six‐dye chemistry analyzed using a novel microfluidic electrophoresis instrument capable of simultaneous detection and discrimination of 8 or more fluorescent dyes. The 27‐locus multiplex offers the potential for a new international STR standard permitting laboratories in any jurisdiction to use a single reaction to determine profiles for loci they typically generate plus an expanded common STR profiling set of global interest.     

Development of a Real‐Time PCR‐Based Method for Analyzing Semen‐Specific Unmethylated DNA Regions and Methylation Status in Aged Body Fluid Stains

The detection of semen in forensic investigation is considered important evidence of sexual assault. In this study, we report the development of a real‐time polymerase chain reaction‐based method for identifying semen that can simply and rapidly analyze the semen‐specific unmethylated region of the DACT1 gene. Using two fluorescent probes designed for the methylated or unmethylated status, this method could perform quantitative analysis of the methylation status in this region. Furthermore, this method was used to analyze various body fluid samples, including 29‐year‐old semen and blood stains. The results showed that this method can detect almost exclusively semen or nonsemen signals even in highly decomposed samples, while a few semen or nonsemen samples showed slight signals of the other fluorescence probe. Although there is still a need for further analysis such as setting thresholds to analyze unknown samples, this method could be a useful supplementary tool for identifying semen, especially in old stains such as those in cold‐case investigations.     

Development of a PCR High‐Resolution Melt Assay for Artemisia absinthium (Wormwood) and a Triplex Assay with Two Additional “Unregulated Legal High” Species Datura stramonium (Jimson Weed) and Merremia tuberosa (Hawaiian Woodrose),

Artemisia absinthium (wormwood), a common ingredient in absinthe, contains the compound thujone, which is unregulated by the U.S. Drug Enforcement Agency. Thujone can cause an “unregulated legal high” in higher concentrations. The European Union limits thujone from Artemisia species to 35 mg/kg while the U.S. Food and Drug Administration requires less than 10 ppm to be “thujone‐free.” However, individuals can smoke or ingest A. absinthium in different forms. This study developed a polymerase chain reaction (PCR) high‐resolution melt (HRM) assay to detect and identify A. absinthium based on primer specificity, sensitivity, repeatability, and robustness. A triplex assay was performed with three “unregulated legal high” species: Datura stramonium, Merremia tuberosa, and A. absinthium; the PCR HRM assay detected and identified each plant at melt temperatures 77.42 ± 0.20°C, 83.88 ± 0.22°C, and 87.77 ± 0.15°C, respectively. The primer set developed distinguished A. absinthium from a variety of plant species and was successfully triplexed.