Results of applying molecular-and-genetic markers of chromosomal DNA in identification of unrecognizable remains of servicemen lost in military actions in the Northern Caucasus

The results of applying molecular-and-genetic markers of chromosomal DNA in identifying unrecognizable remains of servicemen lost as dead in 1994-1996 and 1999-2001 military campaigns in the Northern Caucuses are summarized in the paper. Some of the specific features related with enzymatic amplification typing of DNA preparations sampled from degraded biological tissues of strongly deformed or decayed cadavers were analyzed. The typing results were analyzed by the AB0 system of degraded expertise samples in order to check the reliability of routine forensic-biological examinations as applicable to cadaver tissues with pronounced putrefactive changes. It was established that group-specific antigens of the AB0 system were correctly determined only in 67.5% of cases. False results were obtained in the other 32.5% of cases. Most of them were related with determination of groups 0(I) and A(II).     

An analysis of data curated from 5 years of identifying human remains

An archive of 5 years of cases involving the identification of human remains was curated, collecting information on: The sample type submitted, the number of STR loci yielding interpretable results, the kinship challenge posed, and the outcome for the case. A total of 129 cases of remains ID were investigated using manual DNA extraction and recovery methods with amplification of STR markers using the Power Plex 21 multiplex STR kit from Promega Corp. In 52 cases, blood spots collected by the ME were provided as sample and in 100% of those cases, probabilities of relatedness to the reference samples was ≥99%. In 77 cases, tissue other than blood was provided as a source of DNA. These other samples were grouped categorically into long bones (femur and tibia; 40 cases), skull bones/teeth (11 cases), other bones (16 cases), and tissue (normally adherent to bone) (10 cases). Reference samples provided for cases included alleged parents or child(ren) of the victim (86 cases), alleged full siblings of the victim (38 cases), or alleged second-order relatives (five cases). The overall success rate in confirming the identity of the source of the remains in these cases was 89.2%. Our results demonstrate that a laboratory can be often successful identifying human remains using methods easily implemented in any DNA typing laboratory.     

Internal Validation of Human Mitochondrial DNA Quantification Using Real‐Time PCR

The quantity of mitochondrial DNA (mtDNA) template added for amplification and subsequent dye terminator reactions is critical for obtaining quality sequence data. Validation of a human mtDNA real‐time quantitative PCR (qPCR) assay demonstrated its high degree of reproducibility and precision as well as an extremely sensitive threshold of detection (0.0001 pg/μL or approximately six human mtDNA copies/μL). A study of 35 nonprobative bone and teeth evidence samples revealed that 20 pg of mtDNA template is recommended for successful HV1 and HV2 sequence analysis; however, as little as 0.013 pg can generate a full mtDNA profile when using enhanced amplification reactions. The assay can also detect PCR inhibition and is useful for identifying samples that may benefit from re‐purification. Overall, the assay is an excellent method to quantify mtDNA and is useful for determining the best analytical approach for successful sequencing.     

Typing of deoxyribonucleic acid (DNA) extracted from compact bone from human remains.

The application of deoxyribonucleic acid (DNA) typing methods for the potential identification of unknown human remains was investigated. DNA was isolated from compact bone tissue from badly decomposed bodies and from known and unknown human remains, using a decalcification and ion wash procedure. Restriction fragment length polymorphism (RFLP) analysis of variable number of tandem repeats (VNTR) loci yielded results in some cases, but more often the DNA was too degraded to produce RFLP patterns. No RFLP profiles could be obtained from putrefied soft tissues. However, DNA extracted from compact bone tissue of human remains up to eleven years old was successfully amplified using the polymerase chain reaction (PCR) for the VNTR loci D1S80, D17S5, COL2A1, and APO B, as well as the HLA-DQ alpha locus. This is especially significant, since PCR results were obtained from those samples whose DNA had been degraded substantially and had yielded no RFLP patterns. All DNA types determined from the compact bone tissue from decomposed bodies whose identification had been established first by other means (and whose parents or offspring were available for typing) demonstrated mendelian inheritance of the alleles of the loci analyzed. These results suggest that amplification and typing of DNA extracted from compact bone of human remains could be useful in establishing the identity of a person, as well as in excluding possible false identifications.     

Evaluation of real-time PCR amplification efficiencies to detect PCR inhibitors

Real-time PCR analysis is a sensitive template DNA quantitation strategy that has recently gained considerable attention in the forensic community. However, the utility of real-time PCR methods extends beyond quantitation and allows for simultaneous evaluation of template DNA extraction quality. This study presents a computational method that allows analysts to identify problematic samples with statistical reliability by comparing the amplification efficiencies of unknown template DNA samples with clean standards. In this study, assays with varying concentrations of tannic acid are used to evaluate and adjust sample-specific amplification efficiency calculation methods in order to optimize their inhibitor detection capabilities. Kinetic outlier detection and prediction boundaries are calculated to identify amplification efficiency outliers. Sample-specific amplification efficiencies calculated over a four-cycle interval starting at the threshold cycle can be used to detect reliably the presence of 0.4 ng of tannic acid in a 25 microL PCR reaction. This approach provides analysts with a precise measure of inhibition severity when template samples are compromised. Early detection of problematic samples allows analysts the opportunity to consider inhibitor mitigation strategies prior to genotype or DNA sequence analysis, thereby facilitating sample processing in high-throughput forensic operations.     

Usefulness of dura mater in providing DNA samples for identifying cadavers

We examined the usefulness of the dura mater in identifying human remains. Dura mater was collected from 50 cadavers, including drowned, charred, and mummified remains. The STR genotype using the AmpFlSTR Identifiler Kit could be typed at 15 STR and amelogenin loci in 30 samples of 33 cases. Furthermore, the ABO genotype and amelogenin using gel-based methods could be typed in 44 samples of 50 cases. In cases with successful typing of STR, ABO-DNA, and amelogenin, the longest time after death was from 12 to 26 days in a drowned body. The minimum quantity of dura mater required for DNA extraction was about 2.5 mg, dried and fixed by ethanol, in a cadaver 15 h after death. The state of the DNA from the dura mater from the calvaria may be better than that from the basis cranii interna. We found that DNA from dura mater is one of the most useful samples for forensic identification.     

A novel method for STR-based DNA profiling using microarrays

We describe a novel method for rapidly identifying and distinguishing between different DNA sequences using short tandem repeat (STR) analysis and DNA microarrays. The method can be used to deduce identity, length, and number of STRs of the target molecule. We refer to this technique as the "variable-length probe array" method for STR profiling (VLPA). The method involves hybridization of the unknown STR target sequence to a DNA microarray displaying complementary probes that vary in length to cover the range of possible STRs. A post-hybridization enzymatic digestion of the DNA hybrids is then used to selectively remove labeled single-stranded regions of DNA from the microarray surface. The number of repeats in the unknown target is then deduced based on the pattern of target DNA that remains hybridized to the array. This DNA profiling technique is useful for performing forensic analysis to uniquely identify individual humans or other species.     

Improved MtDNA sequence analysis of forensic remains using a "mini-primer set" amplification strategy

Mitochondrial DNA (mtDNA) analysis of highly degraded skeletal remains is often used for forensic identification due largely to the high genome copy number per cell. Literature from the "ancient DNA" field has shown that highly degraded samples contain populations of intact DNA molecules that are severely restricted in size (1-4). Hand et al. have demonstrated the targeting and preferential amplification of authentic human DNA sequences with small amplicon products of 150 bp or less (1,2). Given this understanding of ancient DNA preservation and amplification, we report an improved approach to forensic mtDNA analysis of hypervariable regions 1 and 2 (HV1/HV2) in highly degraded specimens. This "mini-primer set" (MPS) amplification strategy consists of four overlapping products that span each of the HV regions and range from 126 to 170 bp, with an average size of 141 bp. For this study, 11 extracts representing a range of sample quality were prepared from nonprobative forensic specimens. We demonstrate a significant increase in MPS amplification success when compared to testing methods using approximately 250 bp amplicons. Further, 16 of 17 independent amplifications previously "unreported" due to mixed sequences provided potentially reportable sequence data from a single, authentic template with MPS testing.     

Isolation of DNA from saliva of betel quid chewers using treated cards

Betel quid (BQ) chewing, a common tradition in tropical areas, often poses a problem during collection and DNA analysis of buccal samples from many indigenous communities for population genetic studies and in forensic analysis of chewed BQ residues. This study evaluated the use of FTA card, a chemically treated filter paper, in collecting buccal samples from long-term BQ users and subsequent PCR-based analysis using nine STR markers. A low overall success rate of amplification was observed in the samples extracted using a standard organic extraction procedure (7%) as compared with those prepared using the FTA card (89%). The presence of inhibitors in liquid DNA samples was verified when control DNA failed to amplify in the presence of an equal volume of liquid BQ samples. The use of the FTA card is more practical during field sampling than handling tubes containing buccal swabs.     

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.     

Field Contamination of Skeletonized Human Remains with Exogenous DNA

The Armed Forces DNA Identification Laboratory reports the mitochondrial DNA (mtDNA) sequences of over 800 skeletal samples a year for the Joint POW/MIA Accounting Command–Central Identification Laboratory. These sequences are generated from degraded skeletal remains that are presumed to belong to U.S. service members missing from past military conflicts. In the laboratory, it is possible to control for contamination of remains; however, in the field, it can be difficult to prevent modern DNA from being transferred to skeletal elements and being carried forward through the analysis process. Four such cases are described here along with the controls in place in the laboratory to eliminate the possibility of the exogenous DNA being reported as authentic. In each case, the controls implemented by the laboratories prevented the false reporting of contaminant exogenous DNA from remains that were either faunal or human, but lacked endogenous DNA.     

STR Profiles from DNA Samples with "Undetected" or Low Quantifiler™ Results

Abstract:  Screening methods capable of identifying DNA samples that will not yield short tandem repeat (STR) profiles are desired. In the past, quantitation methods have not been sensitive enough for this purpose. In this study, low level DNA samples were used to assess whether Quantifiler™ has a minimum quantitation value below which STR profiles would consistently fail to be detected. Buccal swabs were obtained and the DNA extracted, quantified, and serially diluted to concentrations ranging from 0.002 to 0.250 ng/μL. Samples were analyzed once with Quantifiler™, followed by Profiler Plus™ amplification and capillary electrophoresis analysis. An absolute minimum value below which STR results were unobtainable could not be defined. From the 96 low level samples tested, STR loci (including one full profile) were successfully amplified and detected from 27% of the samples "undetected" by Quantifiler™. However, no STR alleles were detected in 73% of these "undetected" samples, indicating that Quantifiler™ data may be useful for predicting STR typing success.     

Sex determination by PCR analysis of DNA extracted from incinerated, deciduous teeth.

Establishing the biological sex of human remains is a very important part of identifying victims of fire when severe soft tissue destruction has occurred. Deciduous (children's) teeth were exposed to a range of incineration temperatures 100-500 degrees C for 15 minutes. Polymerase Chain Reaction (PCR) amplification was used to identify specific human amelogenin regions. There was successful identification of human biological sex, from deciduous teeth exposed to incineration temperatures of 200 degrees C and below, using standard ethidium bromide gel staining. There was greater sensitivity using fragment analysis by laser induced fluorescence which achieved sex identification from some teeth heated to 400 degrees C.     

Modified Midi‐ and Mini‐Multiplex PCR Systems for Mitochondrial DNA Control Region Sequence Analysis in Degraded Samples

Two multiplex polymerase chain reaction (PCR) systems (Midiplex and Miniplex) were developed for the amplification of the mitochondrial DNA (mtDNA) control region, and the efficiencies of the multiplexes for amplifying degraded DNA were validated using old skeletal remains. The Midiplex system consisted of two multiplex PCRs to amplify six overlapping amplicons ranging in length from 227 to 267 bp. The Miniplex system consisted of three multiplex PCRs to amplify 10 overlapping short amplicons ranging in length from 142 to 185 bp. Most mtDNA control region sequences of several 60‐year‐old and 400–500‐year‐old skeletal remains were successfully obtained using both PCR systems and consistent with those previously obtained by monoplex amplification. The multiplex system consisting of smaller amplicons is effective for mtDNA sequence analyses of ancient and forensic degraded samples, saving time, cost, and the amount of DNA sample consumed during analysis.     

Development of a one-tube extraction and amplification method for DNA analysis of sperm and epithelial cells recovered from forensic samples by laser microdissection

Laser microdissection can be used in forensic casework to isolate specific cell types from mixtures of biological samples. Extraction of DNA from selected cells is still required prior to STR amplification. Because of the relatively pristine nature of the recovered cells, laser microdissection is more sensitive than more traditional methods of DNA analysis, theoretically resulting in DNA profiles from less cellular material. A one-tube extraction and amplification method minimises loss of DNA through liquid transfers and reduces the potential for contamination events occurring. In this paper, the development of a one-tube method for the effective extraction of DNA from laser microdissected sperm and epithelial cells is described. The performance of the in-house method was compared to that of a commercial DNA extraction kit for extraction of DNA from sperm and the downstream compatibility with STR amplification was determined for both sperm and epithelial samples. Full Identifiler™ profiles after 28 amplification cycles were obtained from as few as 15 epithelial cells and 30 sperm.     

The reliability of forensic osteology--a case in point. Case study

The medico-legal investigation of skeletons is a trans-disciplinary effort by forensic scientists as well as physical anthropologists. The advent of DNA extraction and amplification from bones and teeth has led to the assumption that morphological assessment of skeletal remains might soon become obsolete. But despite the introduction and success of molecular biology, the analysis of skeletal biology will remain an integral part of the identification process. This is due to the fact, that the skeletal record allows relatively fast and accurate inferences about the identity of the victim. Moreover, a standard biological profile may be established to effectively narrow the police investigator's search parameters. The following study demonstrates how skeletal biology may collaborate in the forensic investigation and support DNA fingerprinting evidence.In this case, the information gained from standard morphological methods about the unknown person's sex, age and heritage immediately led the police to suspect, that the remains were that of a young man from Vietnam, who had been missing for 2.5 years. The investigation then quickly shifted to prove the victim's identity via DNA extraction and mtDNA sequence analysis and biostatistical calculations involving questions of kinship [4].     

Spatial Distributions of Oxygen Stable Isotope Ratios in Tap Water From Mexico for Region of Origin Predictions of Unidentified Border Crossers

One of the many difficulties associated with identifying undocumented border crossers stems from an inability to narrow down the search area for the region of origin and family members to obtain family reference samples for DNA comparison. While the geography of regions of origins is wide, the biological profiles of the undocumented border crossers often show strong similarities, young and male. The isotopic composition of human bones, teeth, and hair has been demonstrated to be useful biomarkers for tracing locations and movements of individuals and for aiding in the identification of human remains. Data obtained from human remains can be compared to and aligned with various reference sources, such as soils and bedrock, archaeological remains, or cultural data. Here, the spatial deficiency in isotopic reference data for Mexico, specifically for oxygen (δ¹⁸O) isotopes in tap water, is being addressed through the collection and analysis of over 150 water samples and explored with tooth enamel isotopic values from recently identified Mexican nationals. The isoscape was developed using a Spine with Barriers interpolation method and shows sufficient detail to narrow down the regions where specific isotopic values are represented. The individuals were plotted on the developed isoscape using conditional correlations. The methodology was successful in assigning the correct regions of origin for the two individuals, which shows the tremendous potential of the developed isoscape. Nonetheless, there is more research needed to further improve upon this geolocation method, including analysis of multiple isotopes in different tissues, and the development of new isotopic methods.     

Next generation sequencing technology in Second World War victim identification

The killings during the Second World War (WWII), with nearly 100,000 victims, is one of the greatest losses of life in Slovenia’s modern history and most of the victims are still buried in hidden mass graves and remain unidentified. Identity, ancestry, and phenotypic SNPs, as well as STR markers are already used for solving various cases with Next Generation Sequencing (NGS) technology. In this study, the Precision ID GlobalFiler NGS STR panel was used to identify the WWII victim that could not be identified with capillary electrophoresis (CE) analyses because limited statistical support was obtained after amplification of autosomal STRs using CE STR kits. Bones and teeth were analysed and compared to family references (nephew and niece on paternal line). Prior to DNA isolation 0.5 g of powder was decalcified. The DNA was purified in a Biorobot EZ1 device. The nuclear DNA of the samples was quantified with the PowerQuant kit. Because the recommended posterior probability (PP) of 99.9% was followed with the goal of high confidence of correct identification, the NGS STR Panel was used, and after the analysis of additional STR loci the statistical calculation showed a PP of 99.99986%, showing that a large enough number of genetic markers were analysed when identifying the skeletal remains of the aunt. PP value endorsed the hypothesis that the tooth and bone samples were from individual related to the family references rather than from unrelated individual. In presented case, NGS technology proved to be a powerful tool for increasing the number of autosomal STRs needed for identification of WWII victims when linear markers cannot be used for comparison and only distant relatives are available for analyses.     

Investigation into "normal" background DNA on adult necks: implications for DNA profiling of manual strangulation victims

Others have investigated the role that DNA profiling could play as a method for identifying the perpetrator of manual strangulation. These studies have demonstrated that it is possible to collect offender DNA from the skin surface of a victim following physical contact. It is not known whether nonself biological material is normally present on the skin surface due to adventitious transfer occurring during innocent everyday interactions. To test the hypothesis that detectable amounts of nonself DNA are normally present on the skin surface of healthy adult individuals due to the adventitious transfer of DNA occurring during normal day-to-day social interactions, we designed an experiment in three phases. Phase 1 was used to deduce which DNA collection, extraction, and amplification methods were suited to investigating this question. During phase 2, the neck surface of 24 healthy adult volunteers was swabbed. DNA was extracted using the QIAamp DNA mini kit and amplified using the SGM Plus PCR amplification kit, using 28 PCR cycles. The work carried out during phase 3 involved a simulated assault to investigate primary and secondary transfer of DNA during physical contact. It was found that 23% of neck areas swabbed during phase 2 of this investigation showed nondonor alleles in the resulting DNA profile, with 5% of areas showing six or more nondonor alleles. The results of phase 3 showed that primary, secondary, and zero transfer of victim and/or offender DNA could be observed after physical contact and that alleles from an unknown source could still be detected in this more controlled experiment. The data presented in this paper demonstrate that DNA profiles generated after swabbing the skin surface of healthy adults can include components of an unknown source, present due to adventitious transfer. These components, if present in large quantities, have the potential to interfere with DNA profile interpretation of swabs taken for the investigation of physical assault by DNA profiling.     

两步扩增法在血痕检验中的应用

通过对PCR扩增环节的改良,进而利用两步扩增法对困难血痕进行处理,成功的获得分型.