New optimized DNA extraction protocol for fingerprints deposited on a special self-adhesive security seal and other latent samples used for human identification

Abstract: Obtaining complete short tandem repeat (STR) profiles from fingerprints containing minimal amounts of DNA, using standard extraction techniques, can be difficult. The aim of this study was to evaluate a new kit, Fingerprint DNA Finder (FDF Kit), recently launched for the extraction of DNA and STR profiling from fingerprints placed on a special device known as Self‐Adhesive Security Seal Sticker^® and other latent fingerprints on forensic evidentiary material like metallic guns. The DNA extraction system is based on a reversal of the silica principle, and all the potential inhibiting substances are retained on the surface of a special adsorbent, while nucleic acids are not bound and remain in solution dramatically improving DNA recovery. DNA yield was quite variable among the samples tested, rendering in most of the cases (>90%) complete STR profiles, free of PCR inhibitors, and devoid of artifacts. Even samples with DNA amount below 100 pg could be successfully analyzed.     

Effectiveness of Coupled Application of AmpFℓSTR Yfiler Kit and Reduced Size Y‐chromosomal Short Tandem Repeat Analysis for Archeological Human Bones

The AmpF?STR Yfiler PCR Amplification (Yfiler) kit continues to be improved for a better analytical efficiency in cases of highly degraded DNA. The authors endeavored to determine whether coupling of the Yfiler kit with supplemental multiplex amplification of some Y‐STR loci is a more efficient analytical mode for poorly preserved human femurs (n = 15) discovered at Korean archeological sites. To reveal locus profiles not easily obtained by Yfiler analysis, custom‐designed primers were adopted for the DYS390, DYS391, DYS392, DYS438, DYS439, and DYS635 loci. The success rate for 16 Y‐STR locus profiles obtained from the 15 femurs was improved from 18.33% (in the use of Yfiler kit only) to 49.17% (the coupled use of Yfiler and custom‐designed primers). In this study, the authors established that the custom‐designed primers offer a markedly improved success rate for obtainment of Y‐STR profiles from degraded aDNA not easily identified by sole use of the Yfiler assay.     

Application of low copy number STR typing to the identification of aged, degraded skeletal remains

Low copy number (LCN) STR typing was successfully applied to four interesting cases during developmental validation of the approach for degraded skeletal remains. Specific questions were addressed in each case, with the acquisition of STR data largely serving as additional confirmatory or investigatory information in any specific situation, and not necessarily providing the definitive evidence to establish identity. The cases involve missing U.S. service members from World War I, World War II, and the Vietnam War. The variety of these cases, in terms of the questions addressed, the age of the remains, and the type of reference material available for comparison, demonstrates the broad utility of LCN STR typing in the identification of degraded skeletal remains from missing persons.     

An Accelerated Analytical Process for the Development of STR Profiles for Casework Samples

Significant efforts are being devoted to the development of methods enabling rapid generation of short tandem repeat (STR) profiles in order to reduce turnaround times for the delivery of human identification results from biological evidence. Some of the proposed solutions are still costly and low throughput. This study describes the optimization of an analytical process enabling the generation of complete STR profiles (single‐source or mixed profiles) for human identification in approximately 5 h. This accelerated process uses currently available reagents and standard laboratory equipment. It includes a 30‐min lysis step, a 27‐min DNA extraction using the Promega Maxwell^®16 System, DNA quantification in <1 h using the Qiagen Investigator^® Quantiplex HYres kit, fast amplification (<26 min) of the loci included in AmpF?STR^® Identifiler^®, and analysis of the profiles on the 3500‐series Genetic Analyzer. This combination of fast individual steps produces high‐quality profiling results and offers a cost‐effective alternative approach to rapid DNA analysis.     

Reduction of Powerplex® Y23 Reaction Volume for Genotyping Buccal Cell Samples on FTATM Cards

PowerPlex^® Y23 is a novel kit for Y‐STR typing that includes new highly discriminating loci. The Israel DNA Database laboratory has recently adopted it for routine Y‐STR analysis. This study examined PCR amplification from 1.2‐mm FTA punch in reduced volumes of 5 and 10 μL. Direct amplification and washing of the FTA punches were examined in different PCR cycle numbers. One short robotically performed wash was found to improve the quality and the percent of profiles obtained. The optimal PCR cycle number was determined for 5 and 10 μL reaction volumes. The percent of obtained profiles, color balance, and reproducibility were examined. High‐quality profiles were achieved in 90% and 88% of the samples amplified in 5 and 10 μL, respectively, in the first attempt. Volume reduction to 5 μL has a vast economic impact especially for DNA database laboratories.     

Autosomal short tandem repeat analysis of ancient DNA by coupled use of mini- and conventional STR kits

Abstract: Multiplex autosomal short tandem repeat (STR) genotyping enables researchers to obtain genetic information from ancient human samples. In this study, we tested newly developed AmpF?STR^® MiniFiler? kit for autosomal STR analysis of ancient DNA (aDNA), using human femurs (n = 8) collected from medieval Korean tombs. After extracting aDNA from the bones, autosomal STR analyses were repeated for each sample using the AmpF?STR^® MiniFiler? and Identifiler? kits. Whereas only 21.87% of larger‐sized loci profiles could be obtained with the Identifiler? kit, 75% of the same loci profiles were determined by MiniFiler? kit analysis. This very successful amplification of large‐sized STR markers from highly degraded aDNA suggests that the MiniFiler? kit could be a useful complement to conventional STR kit analysis of ancient samples.     

The application of miniplex primer sets in the analysis of degraded DNA from human skeletal remains

A new set of multiplexed PCR primers has been applied to the analysis of human skeletal remains to determine their efficacy in analyzing degraded DNA. These primer sets, known as Miniplexes, produce shorter amplicons (50-280 base pairs (bp)) than standard short tandem repeat (STR) kits, but still utilize the 13 CODIS STR loci, providing results that are searchable on national DNA databases. In this study, a set of 31 different human remains were exposed to a variety of environmental conditions, extracted, and amplified with commercial and Miniplex DNA typing kits. The amplification efficiency of the Miniplex sets was then compared with the Promega PowerPlex 16 system. Sixty-four percent of the samples generated full profiles when amplified with the Miniplexes, while only 16% of the samples generated full profiles with the Powerplex 16 kit. Complete profiles were obtained for 11 of the 12 Miniplex loci with amplicon sizes less than 200 bp. These data suggest smaller PCR amplicons may provide a useful alternative to mitochondrial DNA for anthropological and forensic analysis of degraded DNA from human skeletal remains.     

An Optimized DNA Analysis Workflow for the Sampling,Extraction, and Concentration of DNA obtained from Archived Latent Fingerprints

DNA profiles have been obtained from fingerprints, but there is limited knowledge regarding DNA analysis from archived latent fingerprints—touch DNA “sandwiched” between adhesive and paper. Thus, this study sought to comparatively analyze a variety of collection and analytical methods in an effort to seek an optimized workflow for this specific sample type. Untreated and treated archived latent fingerprints were utilized to compare different biological sampling techniques, swab diluents, DNA extraction systems, DNA concentration practices, and post‐amplification purification methods. Archived latent fingerprints disassembled and sampled via direct cutting, followed by DNA extracted using the QIAamp® DNA Investigator Kit, and concentration with Centri‐Sep? columns increased the odds of obtaining an STR profile. Using the recommended DNA workflow, 9 of the 10 samples provided STR profiles, which included 7–100% of the expected STR alleles and two full profiles. Thus, with carefully selected procedures, archived latent fingerprints can be a viable DNA source for criminal investigations including cold/postconviction cases.     

Collecting and Analyzing DNA Evidence from Fingernails: A Comparative Study,,

Forensic practitioners and crime laboratories regularly collect and analyze fingernail evidence; however, the best techniques for processing such evidence have not been established. In this study, numerous aspects of fingernail evidence processing—collection of exogenous cells, transportation, purification of DNA, and STR analysis—were analyzed using fingernails harboring applied blood or epithelial cells from scratchings. Autosomal STR mixtures resulted when fingernails were soaked or swabbed, while scrapings rarely generated mixtures but exhibited allelic dropout. Y‐STRs yielded single source profiles, with scrapings again showing dropout. A silica‐based kit extraction recovered significantly more exogenous DNA than did organic extraction, neither of which was affected by nail polish. Swabbing nails in succession resulted in some cross‐contamination from exogenous material, while transporting nails together did not, although there was loss of exogenous cells. Optimized nail processing produced complete Y‐STR profiles of male volunteers from female fingernails following scratchings.     

A Y‐Chromosomal Haplotype with Two Short Tandem Repeat Mutations*

Abstract:  The male‐specific Y‐chromosomal short tandem repeat (STR) is a useful tool in forensic casework. The Y haplotype comprised of 16 loci, which is amplified simultaneously by AmpFlSTR^® Yfiler^TM PCR kit and provides strong exculpatory evidence in individual identification. We reported a rare Y‐STR profile with a null allele at the DYS448 locus and an off‐ladder allele at the DYS456 locus, when genotyping material from a vaginal swab in an alleged rape case. Sequence analysis revealed that the DYS448 null allele was a true type of null allele because of a total deletion of 11 upstream repeats and 9 bp of the N₄₂ region, and there were numerous primer binding site mutations as well. The amplicon of the DYS456 locus was a small 92‐bp fragment that was off‐ladder, and sequencing analysis showed that there were only 10 repeats (AGAT)₁₀. This Y chromosome haplotype that was comprised of two variations provided helpful evidence for personal identification.     

DNA Profiling Success Rates from Degraded Skeletal Remains in Guatemala

No data are available regarding the success of DNA Short Tandem Repeat (STR) profiling from degraded skeletal remains in Guatemala. Therefore, DNA profiling success rates relating to 2595 skeletons from eleven cases at the Forensic Anthropology Foundation of Guatemala (FAFG) are presented. The typical postmortem interval was 30 years. DNA was extracted from bone powder and amplified using Identifiler and Minifler. DNA profiling success rates differed between cases, ranging from 50.8% to 7.0%, the overall success rate for samples was 36.3%. The best DNA profiling success rates were obtained from femur (36.2%) and tooth (33.7%) samples. DNA profiles were significantly better from lower body bones than upper body bones (p = <0.0001). Bone samples from males gave significantly better profiles than samples from females (p = <0.0001). These results are believed to be related to bone density. The findings are important for designing forensic DNA sampling strategies in future victim recovery investigations.     

GeneMarker® HID: A reliable software tool for the analysis of forensic STR data

Abstract: GeneMarker^® HID was assessed as a software tool for the analysis of forensic short tandem repeat (STR) data and as a resource for analysis of custom STR multiplexes. The software is easy to learn and use, and includes design features that have the potential to reduce user fatigue. To illustrate reliability and accuracy, STR data from both single‐source and mixture profiles were analyzed and compared to profiles interpreted with another software package. A total of 1898 STR profiles representing 28,470 loci and more than 42,000 alleles were analyzed with 100% concordance. GeneMarker HID was also used to successfully analyze data generated from a custom STR multiplex, with simplified and rapid implementation. Finally, the impact of the user‐friendly design features of the software was assessed through a time scale study. The results suggest that laboratories can reduce the time required for data analysis by at least 25% when using GeneMarker HID.     

Multiplex short tandem repeat amplification of low template DNA samples with the addition of proofreading enzymes

Abstract:  With <100 pg of template DNA, routine short tandem repeat (STR) analysis often fails, resulting in no or partial profiles and increased stochastic effects. To overcome this, some have investigated preamplification methods that include the addition of proofreading enzymes to the PCR cocktail. This project sought to determine whether adding proofreading polymerases directly in the STR amplification mixture would improve the reaction when little template DNA is available. Platinum Taq High Fidelity and GeneAmp High Fidelity were tested in Profiler Plus? STR reactions alone and in combination with AmpliTaq^® Gold. All reactions included the additional step of a post‐PCR purification step. With both pristine low template DNA and casework samples, the addition of these polymerases resulted in comparable or no improvement in the STR amplification signal. Further, stochastic effects and artifacts were observed equally across all enzyme conditions. Based on these studies, the addition of these proofreading enzymes to a multiplex STR amplification is not recommended for low template DNA work.     

Identification of two fire victims by comparative nuclear DNA typing of skeletal remains and stored umbilical tissues

We describe here our collaborative efforts in identifying 2 fatalities of a fire disaster by using a variety of identification techniques. Postmortem findings in both cases were reinforced using Short Tandem Repeat (STR) DNA technology to establish with a high degree of certainty the identities of 2 child victims. STR markers used in the present study include HUMAMEL, HUMCSFIPO, HUMTHO1, HUMvWA, HUMFES/FPS, HUMF13A01, HUMFOLP23, D8S3O6, HUMFGA, and HUMTPOX. Unambiguous identification was made possible through matching DNA profiles generated from skeletal remains with those from umbilical tissues. These tissues were kept by their mothers in accordance with a Philippine tradition and were submitted for DNA analysis. Of the DNA profiles generated from exhumed bone samples of 21 child victims, comparison with the genetic profiles of children A and B obtained from umbilical tissues showed consistent DNA matches with remains 1756 and 1758, respectively.     

Genetic Identification of Communist Crimes’ Victims (1944–1956) Based on the Analysis of One of Many Mass Graves Discovered on the Powazki Military Cemetery in Warsaw,Poland

As the result of the communist terror in Poland, during years 1944–1956 more than 50,000 people died. Their bodies were buried secretly, and most places are still unknown. The research presents the results of identification of people buried in one of many mass graves, which were found at the cemetery Pow?zki Military in Warsaw, Poland. Exhumation revealed the remains of eight people, among which seven were identified genetically. Well‐preserved molars were used for the study. Reference material was collected from the closest living relatives. In one case, an exhumation of victim's parents had to be performed. DNA from swabs was extracted with a PrepFiler^® BTA Forensic DNA Extraction Kit and organic method. Autosomal, Y‐STR amplification, and mtDNA sequencing were performed. The biostatistical calculations resulted in LR values from 1608 to 928 × 10¹⁸. So far, remains of more than 50 victims were identified.     

Extraction of DNA from Skeletonized Postcranial Remains: A Discussion of Protocols and Testing Modalities

This paper provides a retrospective of the DNA analysis performed by the Armed Forces Medical Examiner–Armed Forces DNA Identification Laboratory between 1990 and 2018. Over 13,000 postcranial osseous materials, comprised of wartime losses from World War II, the Korean War, and South‐East Asia, were examined by the following: mitochondrial DNA sequencing, a modified AmpFlSTR® Yfiler?, AmpFlSTR® MiniFiler?, PowerPlex® Fusion, or NGS. Four different DNA extraction protocols were used: incomplete demineralization coupled with an organic purification; complete demineralization with an organic purification; complete demineralization with an inorganic purification using QIAquick PCR Purification Kit; and a protocol designed specifically for use with next‐generation sequencing. In general, complete demineralization coupled with an organic purification was the optimal extraction protocol for sequencing of mitochondrial DNA, regardless of the osseous element tested. For STR testing, demineralization paired with an inorganic purification provided optimum results, regardless of kit used or osseous element tested.     

From unknown to known: Identification of the remains at the mausoleum of fosse Ardeatine

During the Second World War, on 24th March 1944, 335 Italians were massacred near Rome by the occupying forces of Nazi Germany. Four months later forensic examination led to the identification of 323 out of 335 victims. After approximately 60?years, the identification of the remaining unidentified twelve victims began with anthropological and genetic analysis carried out by a team of Italian forensic experts. Anthropological analysis was performed in field in order to confirm the sex of each victim and verify the presence of only one individual in each grave for a correct sampling. Selected bone fragments for each individual were then collected and transferred to the laboratory for genetic analysis. Although the anthropological ante mortem information was limited, morphological and metrical data was collected for a possible future identification of the victims. Subsequently, the typing of autosomal loci, Y-STR and mtDNA D-loop region of all bone and available reference samples was conducted. LR and cumulative LRs obtained from autosomal STR and Y-STR results confirmed the alleged relationship between three victims and their relatives with values over 10⁴ (one sample) and 10⁶ (two samples). Therefore, the genetic analysis offered the families the possibility of replacing the number of the grave with the name of the victim.     

Developmental Validation of the PrepFiler™ Forensic DNA Extraction Kit for Extraction of Genomic DNA from Biological Samples*

Abstract: The PrepFiler? Forensic DNA Extraction Kit enables isolation of genomic DNA from a variety of biological samples. The kit facilitates reversible binding of DNA with magnetic particles resulting in high DNA recovery from samples with very low and high quantities of biological materials: 0.1 and 40 μL of human blood (donor 2) provided 14 and 2883 ng of DNA, respectively. Following the revised SWGDAM guidelines, performance of the developed method was investigated using different sample types including saliva on swabs, semen stains on cotton fabric, samples exposed to environment, samples with polymerase chain reaction (PCR) inhibitors, blood stains (on denim, cotton cloth, and FTA^® paper), and touch evidence‐type samples. DNA yields for all samples tested were equal or better than those obtained by both phenol–chloroform extraction and commercial kits tested. DNA obtained from these samples was free of detectable PCR inhibitors. Short tandem repeat profiles were complete, conclusive, and devoid of PCR artifacts.     

The 2018 California Wildfires: Integration of Rapid DNA to Dramatically Accelerate Victim Identification

In November 2018, Butte County, California, was decimated by the Camp Fire, the deadliest wildfire in state history. Over 150,000 acres were destroyed, and at its peak, the fire consumed eighty acres per minute. The speed and intensity of the oncoming flames killed scores of people, and weeks before the fire was contained, first responders began searching through the rubble of 18,804 residences and commercial buildings. As with most mass disasters, conventional identification modalities (e.g., fingerprints, odontology, hardware) were utilized to identify victims. The intensity and duration of the fire severely degraded most of the remains, and these approaches were useful in only 22 of 84 cases. In the past, the remaining cases would have been subjected to conventional DNA analysis, which may have required months to years. Instead, Rapid DNA technology was utilized (in a rented recreational vehicle outside the Sacramento morgue) in the victim identification effort. Sixty-nine sets of remains were subjected to Rapid DNA Identification and, of these, 62 (89.9%) generated short tandem repeat profiles that were subjected to familial searching; essentially all these profiles were produced within hours of sample receipt. Samples successfully utilized for DNA identification included blood, bone, liver, muscle, soft tissue of unknown origin, and brain. In tandem with processing of 255 family reference samples, 58 victims were identified. This work represents the first use of Rapid DNA Identification in a mass casualty event, and the results support the use of Rapid DNA as an integrated tool with conventional disaster victim identification modalities.     

The Effects of Different Maceration Techniques on Nuclear DNA Amplification Using Human Bone

Abstract: Forensic anthropologists routinely macerate human bone for the purposes of identity and trauma analysis, but the heat and chemical treatments used can destroy genetic evidence. As a follow‐up to a previous study on nuclear DNA recovery that used pig ribs, this study utilizes human skeletal remains treated with various bone maceration techniques for nuclear DNA amplification using the standard Combined DNA Index System (CODIS) markers. DNA was extracted from 18 samples of human lower leg bones subjected to nine chemical and heat maceration techniques. Genotyping was carried out using the AmpF?STR^® COfiler^® and AmpF?STR^® Profiler Plus^® ID kits. Results showed that heat treatments via microwave or Biz/Na₂CO₃ in sub‐boiling water efficiently macerate bone and produce amplifiable nuclear DNA for genetic analysis. Long‐term use of chemicals such as hydrogen peroxide is discouraged as it results in poor bone quality and has deleterious effects on DNA amplification.