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.     

Preparation of degraded human DNA under controlled conditions

DNA typing through analysis of short tandem repeats (STRs) and mitochondrial DNA (mtDNA) by means of the polymerase chain reaction (PCR) and sequencing are the common methods for the forensic identification of persons and reconstruction of kinship, especially when skeletal human remains have to be analyzed. Furthermore, samples typically found at crime scenes may be both quantitatively and qualitatively inadequate since they may contain very scarce and often degraded DNA due to exposure to heat, light, humidity, and microorganisms. In order to improve the performance of STR typing technology in those cases where DNA availability is limited, it would be desirable to have a source of degraded DNA with known properties. For this purpose, we have developed a method to prepare artificially degraded DNA under controlled conditions. By treatment of genomic DNA with sonication and DNAse I we have produced DNA fragments within a defined range of lengths. STR typing of this degraded DNA with a commercially available multiplex kit could only produce partial profiles as indicated by the absence of STR alleles with sizes >200 bp. This artificially degraded DNA can be used for the improvement and standardization of STR typing protocols when only highly degraded DNA is available for analysis.     

Quantitative and qualitative analysis of DNA extracted from postmortem muscle tissues

DNA extracted from 33 postmortem muscle specimens was analyzed using MZ 1.3, a hypervariable minisatellite probe, as well as locus-specific minisatellite probes (g3, MS1 and MS43). After storage at -25 degrees C for 10 months, DNA from all the samples was partially (approximately 21% of total DNA) degraded even when autopsy was performed 1 day postmortem. However, more than 90% of DNA samples up to at least 3 days postmortem were suitable to obtain good restriction fragment length polymorphism (RFLP) patterns. When small strips of specimen were stored for 8 days at room temperature in moist chambers, approximately 42% of total DNA was degraded. Only 30% of these DNA samples still showed good RFLP patterns. However, no obvious relation between qualities of DNA analyzed by detection of RFLP and quantities of total and high-MW DNA became apparent. A case of familial relationship was ascertained by DNA fingerprints. Since DNA of good quality can be recovered from muscle tissues in large quantities, DNA extraction from muscle tissues and detection of RFLP patterns should be very useful for individual identification in autopsy cases.     

Analysis of forensic samples in Banco Nacional de Datos Genéticos

Analysis of forensic samples to evaluate the rate of success for molecular markers: autosomal STRs, Y chromosome, and mitochondrial DNA. Since 2006 to date a total of 390 forensic samples were analyzed: bones, teeth, hairs, swabs, stains and paraffin embedded tissue. Bones and teeth, were pulverized in a Freezer Mill, extracted by chloroform/phenol/isoamyl alcohol method, and then purified with Centricon 100 columns. DNA from paraffin was extracted with QIAmp DNA Mini kit (QIAGEN). Mitochondrial DNA Control Region sequences were determined for regions HV1/HV2. Sequencing was performed using the BigDye^® Terminator v 1.1 Kit and analyzed in ABIPRISM^® 3100 Genetic Analyzer (AB). STRs were amplified using Amp FlSTR Identifiler^®, Minifiler^® and YFiler^® Kit (AB) and analyzed in ABI PRISM^® 3100 Genetic Analyzer and ABI PRISM^® 3130xl Genetic Analyzer (AB). Among forensic samples, bones and teeth analyzed for autosomal STRs, we obtained successful results in all of them. Incomplete typing are represented by loci of higher molecular weight, which demonstrates the poor quality of the sample due to its state of degradation and obtained better results using mini STRs. Successful results in sequencing for mitochondrial HV1 region for all samples analyzed, but in few hair samples we obtained mixed sequences and that represented important difficulties for the analysis. Age of samples and conservation are factors related which affect DNA viability. Autosomal STRs solved all the samples analyzed in our study, but Y chromosome analysis and mitochondrial DNA sequencing are also important and necessary markers in some forensic cases.     

The personal identification of many samples recovered from under the sea

An automatic and rapid DNA typing system was employed for personal identification, using fragmentary tissue samples from victims in an airplane accident. Two victims were crushed into small pieces, and 33 samples suspected to belong to them were recovered from under the sea. From each sample, 10 mg was used for testing. The parents' bloods of two presumptive victims were also examined. DNA extraction from samples was performed by the NaI method, and the obtained DNA samples were analyzed with the ABI PRISM system. Among 33 samples, 31 samples were identified to be human tissues, possibly from two victims. The other two samples seemed to be parts of marine animals. ABO blood group, STR polymorphism, and mitochondrial DNA polymorphism typing were possible in every examined human sample. Two victims' fragmentary tissues were identified by determining ABO genotype, STR type and mitochondrial DNA type. The system we employed enabled an accurate typing of many fragmentary samples in a short time, thus contributing to the fast and secure identification of many victims in such cases as big air accidents.     

Quantifiler Human DNA Quantification Kit (Applied Biosystems) as a screening kit for DNA profiling

This study investigates the connection between the results of DNA quantification with Quantifiler Human DNA Quantification Kit (AB) and DNA profiling. For this purpose the DNA concentration of 3.068 routine casework samples was determined and DNA profiling was carried out. For discussion, depending on the specific DNA concentration, the samples were divided into four groups (0–5, 5–10, 10–30 and more then 30 pg/μl DNA) and the obtained number of full or partial profiles and the negative typing results was listed. Moreover group 1 (0–5 pg/μl DNA) results were subdivided and analysed more precisely. Based on the amount of 4% positive typing results in group 1 we decided to analyse every sample with quantification results >0 pg/μl DNA. A real cut-off no longer exists. Only samples showing 0 pg/μl on each result were sorted out.     

Basic issues in forensic DNA typing

DNA analysis has become the standard method in forensic stain typing (termed DNA profiling). In contrast to conventional serological methods, any human tissue or body fluid can be analysed by DNA profiling as long as it contains nucleated cells. The majority of genetic systems studied at the DNA level are derived from “non-coding” portions from the human genome, and are located either in the vicinity of expressed (coding) genes or in stretches of DNA sequences interspersing with the genes. The typing results are usually recorded as DNA fragment lengths or “alleles” indicating the number of core repeat elements for short tandem repeat systems. These typing results do not contain any useful information which might reveal genetic traits or predispositions for inherited disease about the individual studied. Typing systems for DNA profiling are predominantly selected according to criteria related to the robustness for typing of (potentially degraded) forensic specimens, the degree of genetic polymorphism (which influences the chance to exclude a wrongfully accused person), and the amenability to standardisation as a basis to obtain reproducible results.     

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.     

Typing of STR locus in biological objects with pronounced degeneration: analysis of DNA in exhumed remains of victims of war actions

The authors describe some specific features of enzymatic amplification typing of DNA preparations obtained from degraded tissues of remains of humans, which were brought from regions of war actions in the Chechen Republic. Special attention is paid to the specific artefact of polymerase chain reaction, for the first time detected by the authors in examinations of the above-mentioned objects. This so-called "ladder" effect manifesting by simultaneous nonspecific amplification of many variants of allele fragments of the STR locus on the individual DNA matrix, which can be erroneously interpreted as an evidence of mixed DNA preparation, is apparently characteristic of individual objects with pronounced degradation of biological material. Such phenomena were observed in typing of STR locuses in biological tissues subjected to biological, thermal, and physicohemical degradation. The phenomenon was studied in detail.     

Assessment of STR Typing Success Rate in Soft Tissues from Putrefied Bodies Based on a Quantitative Grading System for Putrefaction

To date, there is no systematic investigation of the association of short tandem repeat (STR) typing success rate in soft tissues with different signs of putrefaction. Herein, putrefaction was rated using a newly developed 19‐parameter system in soft tissues from a collective of 68 decaying bodies, and DNA yield was determined in 408 samples. DNA integrity was rated using a self‐devised pentaplex PCR generating an “integrity score” (S_i). STR typing success rate was then assessed for selected cases. DNA yield and S_i differed significantly between tissues with kidney on average exhibiting the highest S_i values. Statistical analysis revealed that nine parameters were significantly and positively correlated with S_i. The observed values for each of these nine parameters were summed up to generate a putrefaction score (S_p) for each sample. Our results show that STR typing success rate can be predicted based on S_p before expensive multiplex STR profiling is performed.     

The effectiveness of various strategies to improve DNA analysis of formaldehyde-damaged tissues from embalmed cadavers for human identification purposes

Formalin-fixed tissues provide the medical and forensic communities with alternative and often last resort sources of DNA for identification or diagnostic purposes. The DNA in these samples can be highly degraded and chemically damaged, making downstream genotyping using short tandem repeats (STRs) challenging. Therefore, the use of alternative genetic markers, methods that pre-amplify the low amount of good quality DNA present, or methods that repair the damaged DNA template may provide more probative genetic information. This study investigated whether whole genome amplification (WGA) and DNA repair could improve STR typing of formaldehyde-damaged (FD) tissues from embalmed cadavers. Additionally, comparative genotyping success using bi-allelic markers, including INDELs and SNPs, was explored. Calculated random match probabilities (RMPs) using traditional STRs, INDEL markers, and two next generation sequencing (NGS) panels were compared across all samples. Overall, results showed that neither WGA nor DNA repair substantially improved STR success rates from formalin-fixed tissue samples. However, when DNA from FD samples was genotyped using INDEL and SNP-based panels, the RMP of each sample was markedly lower than the RMPs calculated from partial STR profiles. Therefore, the results of this study suggest that rather than attempting to improve the quantity and quality of severely damaged and degraded DNA prior to STR typing, a more productive approach may be to target smaller amplicons to provide more discriminatory DNA identifications. Furthermore, an NGS panel with less loci may yield better results when examining FD samples, due to more optimized chemistries that result in greater allelic balance and amplicon coverage.     

miniSTR荧光检测体系的建立及法医学应用

目的 建立检测片段均小于150bp的miniSTR荧光检测体系,提高对微量降解检材DNA的检测效能. 方法 应用Primer Premier 5软件设计、FastPCR 6.0筛选引物,组合成用四色荧光标记引物的miniSTR复合扩增体系.优化PCR检测条件和引物浓度,在3100-Avant仪上用POP4胶进行电泳检测.分型结果用DNA标准品9947A和007进行验证,并通过检测新鲜血样、疑难微量检材评估该体系的法医学应用效能.结果 建立的miniSTR荧光检测体系（D12A TA 63、D2S1776、D1GA TA 113、D4S2408、D17S974、D20S482、D3S3053、Ame logenin、D6S474、D9S1122）中各基因座的检测片段均小于150bp,各等位基因扩增均衡性良好,无非特异性扩增产物,等位基因频率分布符合Hardy-Weinberg平衡,累积个体识别率为0.999999983,三联体累积非父排除率为0.996 8.能成功检测腐败肌肉组织、低拷贝数DNA检材以及在40％甲醛溶液中固定12d的人体组织. 结论 miniSTR荧光检测体系可独立应用于降解DNA样本的个体识别鉴定或补充应用于亲权鉴定,提高对微量、降解检材DNA的检测能力.     

荧光标记短片段STR复合扩增系统的法医学应用研究

目的解决严重降解(低于300bp)DNA检验问题,提高降解DNA的检出率。方法重新设计引物,减小扩增产物片段长度,用荧光标记短片段STR复合扩增体系进行DNA检验,扩增结果与用Identifiler试剂盒扩增出的结果进行比较。结果用荧光标记短片段STR复合扩增系统对实际案件中的高度降解DNA检材进行检验,可以获得满意分型。结论该系统可用于严重降解DNA检材的检验工作。     

Study of human salivary agglutinins in expert evaluation of disputable paternity

Analysis of the saliva for evaluation of AB0 production should be carried out along with other studies in expert evaluation of disputable paternity and maternity and in cases with replacement of children. A total of 249 subjects (79 families) were examined. Salivary specimens from 72 families were analyzed; salivary specimens were not analyzed in 7 families in which both men and women had blood group IV (AB0) and no agglutinins were present in the serum. The production of isoantibodies alpha and beta is inherited similarly as the production of AB0 isoantigens. The majority (81.6%) of children whose parents were isoantibody producers, were isoantibody producers too. If one parent produced isoantibodies and the other not, the children were more often (68.1%) producers, but less often than in the previous category. If both parents were non-producers, the children were non-producers as well.     

Analysis of mitochondrial SNPs in addition to conventional STR-typing in a case of aggravated theft

In the field of forensic DNA typing, the analysis of Short Tandem Repeats (STRs) can fail in cases of degraded DNA. The typing of coding region Single Nucleotide Polymorphisms (SNPs) of the mitochondrial genome provides an approach to acquire additional information. In the examined case of aggravated theft, both suspects could be excluded of having left the analyzed hair on the crime scene by SNP typing. This conclusion was not possible subsequent to STR typing. SNP typing of the trace on the torch light left on the crime scene increased the likelihood for suspect no. 2 to be the origin of this trace. This finding was already indicated by STR analysis. Suspect no. 1 was excluded for being the origin of this trace by SNP typing which was also indicated by STR analysis. A limiting factor for the analysis of SNPs is the maternal inheritance of mitochondrial DNA. Individualisation is not possible. In conclusion, it can be said that in the case of traces which cause problems with conventional STR typing the supplementary analysis of coding region SNPs from the mitochondrial genome is very reasonable and greatly contributes to the refinement of analysis methods in the field of forensic genetics.     

Development of a forensic DNA phenotyping panel using massive parallel sequencing

The HIrisPlex-S system, targeting a total of 41 SNPs, allows the simultaneous eye, hair and skin color prediction from DNA. In the present study, we developed a massive parallel sequencing (MPS) multiplex assay in order to genotype all the HIrisPlex-S markers in degraded casework samples. PCR amplicons sizes of target regions were kept below 180 bp, in order to allow analysis of degraded DNA samples. Individuals with known phenotype, artificially degraded DNA samples and a set of 2800M control DNA dilutions were sequenced on a Ion PGM System, in order to evaluate the concordance testing results and the forensic suitability of this 41-plex MPS assay. Full and reliable profiles could be obtained with 0.1 ng of input DNA. The increment of the number of PCR cycles results in improvement of sensitivity or in typing results but an increase of artifacts were also observed.     

法医SNP复合检测体系的构建及应用

目的构建48-SNP位点复合检测体系,用于个体识别、性别鉴定、ABO基因分型。方法采集225份无关个体样本(血斑及口腔拭子),18份案例样本(不同组织及体液斑),选择43个常染色体位点、4个ABO基因位点和1个性别鉴定位点,根据单碱基延伸技术通过GenomeLabTMSNPstream基因分型系统进行SNP分型;并检测体系灵敏度、同一个体不同组织同一性及模拟腐败检材。结果 48-SNP体系分型结果与测序结果的一致性为100%,最小DNA检出量为0.25ng,不同组织来源样本检测同一性很好;利用该体系检测225名无关汉族个体,所有位点均符合Hardy-Weinberg平衡,整个系统的随机匹配概率为9.4×10-18,累积非父排除率(CEP)为0.999 788,累积个体识别率大于0.999 999 999 999 999 99。结论本文48-SNP体系能同时进行个体识别、ABO基因分型和性别鉴定,可以作为现有STR检验体系的补充。     

Heteroplasmy in hair: differences among hair and blood from the same individuals are still a matter of debate

The analysis of mitochondrial DNA (mtDNA) is a useful tool in forensic cases when sample contents too little or degraded nuclear DNA to genotype by autosomal short tandem repeat (STR) loci, but it is especially useful when the only forensic evidence is a hair shaft. Several authors have related differences in mtDNA from different tissues within the same individual, with high frequency of heteroplasmic variants in hair, as also in some other tissues. Is still a matter of debate how the differences influence the interpretation forensic protocols. One difference between two samples supposed to be originated from the same individual are related to an inconclusive result, but depending on the tissue and the position of the difference it should have a different interpretation, based on mutation-rate heterogeneity of mtDNA. In order to investigate it differences in the mtDNA control region from hair shafts and blood in our population, sequences from the hypervariable regions 1 and 2 (HV1 and HV2) from 100 Brazilian unrelated individuals were compared. The frequency of point heteroplasmy observed in hair was 10.5% by sequencing. Our study confirms the results related by other authors that concluded that small differences within tissues should be interpreted with caution especially when analyzing hair samples.     

Persisting fetal microchimerism does not interfere with forensic Y-chromosome typing

Forensic Y-chromosome typing applies Y-chromosomal polymorphisms to the analysis of male/female mixed stains such as vaginal swabs in rape cases. The sensitivity of this approach exceeds that of cytological techniques combined with autosomal DNA typing. Y-chromosome typing is based on the assumption that Y-chromosomal DNA found in tissue or secretions of women must originate from a male individual, usually the perpetrator. Nevertheless, it was shown recently that fetal cells can migrate into the female body during pregnancy and can persist for decades ("persisting fetal microchimerism"). The body of a woman after a pregnancy with a male embryo can thus display a small fraction of fetal cells with Y-chromosomes. Using high sensitivity PCR protocols (reamplification with nested primers and up to 60 PCR cycles) fetal cells were previously identified in a number of maternal tissues including skin, blood, muscle and solid organs. It is, however, not clear at present, whether these cells can occur in vaginal secretions, and whether they are capable of producing false positive results in forensic Y-chromosome typing. To evaluate these questions, 66 blood samples of women with at least one son and nine vaginal swabs of women without sexual intercourse in the last 2 weeks were amplified for a stretch of the SRY gene. Eight thyroid gland tissues with already established male fetal microchimerism were used as positive control samples. Blood samples of 10 young girls without history of pregnancy were used as negative controls. Using a PCR with 10 ng of extracted DNA and 30 PCR cycles ("routine sensitivity assay") none of the samples yielded positive results. However, in a PCR with 200 ng of extracted DNA and 45 PCR cycles ("high sensibility assay"), 14% of the blood samples of mothers and 33% of the vaginal swabs amplified for SRY. Our results thus show that increasing the sensitivity of the PCR method and the amount of template DNA produce positive results while protocols used for routine Y-chromosomal typing with small amounts of DNA (approximately 10 ng of DNA) and with a limited number of PCR cycles (approximately 30) can clearly eliminate this peril.     

Significance of mitochondrial DNA for forensic stain analysis, identification and forensic lineage determination

DNA testing using conventional STR systems may produce insufficient results, if the genomic DNA in the specimen is either highly degraded or the available quantity is very small (e.g. skin particles, hair shafts or ancient bones). In some of these cases the examination of mitochondrial DNA, which is present in considerably larger copy numbers in the cytoplasm, is more successful than that of nuclear DNA. Identification of unknown corpses by conventional DNA typing sometimes remains doubtful, if only samples from presumably distant relatives or putative brothers or sisters are available for comparison. Since mitochondrial DNA is generally transmitted in maternal lineages, its sequence pattern can be directly compared with those of other individuals and, in case of the same maternal lineage, corresponding sequence chromatograms are to be expected. In connection with nuclear DNA typing methods certain sequence motives may furnish clues to ethnic groups. The report presents three cases illustrating the application possibilities of mtDNA typing in forensic practice.