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).     

Development and application of hydrogel oligonucleotide microchips for forensic-medical personal identification as illustrated by ABO locus

The article describes the method defining 5 alleles of ABO blood group typing system by molecular hybridization in hydrogel oligonucleotide microchip. The testing points were SNP variants in positions 261 and 297of exon 6 and in positions 646 and 657 of exon 7. Therefore, 5 ABO blood groups can be easily revealed: A, B, 0(1), 0(1v), 0(2). The method was tested on 10 DNA samples isolated from blood and saliva of unrelated individuals. The results were confirmed by sequencing of the identified allelic fragments. Estimation sensitivity was 25 pg of total DNA input. This technique is cost-effective and easy for use and, therefore, promising for forensic-medical personal identification.     

Use of amelogenin test for DNA sex appurtenance in analysis of mixed biological traces

Theoretical and experimental study of amelogenin tests system for identification of DNA sex appurtenance has been performed. In many cases amplification can fail to reflect the true quantitative ratio between the mixture components during typing sex-specific variants of amelogenin gene in DNA preparations of mixed sex appurtenance. In practical studies these properties of the amelogenin system can lead to difficulties and errors in interpretation of the results of forensic medical expert evaluations, and we therefore studied the use of amelogenin DNA test for identification of sex appurtenance of mixed biological traces and compared the characteristics of autosomal PDAF system and amelogenin system for sex identification in analysis of DNA mixtures.     

Optimization of the biological microchip for genotyping the AB0 locus: analytical aspects

The present work continues the search for methodological options facilitating the improvement and optimization of the biological microchip designed for genotyping the AB0 locus. It was shown in an earlier study designed to test a prototype biological microchip using a reference set of preparations with the known group specificity that under certain conditions some cells of the biochip appear to generate artifact hybridization signals that tend to make the results of genotyping either incorrect or difficult to interpret. We performed the correction of the molecular structure of DNA probes of the prototype biochip for the purpose of optimization of their hybridization potency. In addition, we developed and synthesized new DNA probes and designed new variants of the biochip. The experimental analysis of hybridization properties of all DNA probes thus obtained was carried out for the final choice of the most promising options suitable for the creation of the optimized biochip.     

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.     

The restrictase analysis of human DNA as a method for determining genetic sex in forensic biological expertise]

In this article phenomena related to sex heteromorphism of restrictase hydrolysates of DNA, isolated from objects of expert analysis was recommended for use. The performed investigations allow one to work out the system of discriminating sex of biological objects, based on restrictase analysis of human DNA and on registration of sex-specific restrictase fragments. Possibilities of method and its value for gene-identification expertise were illustrated using certain cases from expert practice.     

Transfer of biological traces in cases of hanging and ligature strangulation

In hanging and ligature strangulation, the noose mostly causes a mark or groove which is formed partly by compression of the skin and partly by abrasion with loss of the upper epidermal layers. The horny scales abraded from the neck may be transferred to the strangulation device or to the interposed textiles where they are sometimes visible at stereomicroscopic examination or even to the naked eye as silver-grey particles. The morphologic features of the epidermal transfer due to hanging and ligature strangulation is demonstrated by 14 case examples. The biological traces may be sufficient for comparative DNA typing by means of PCR-based methods. In 9 out of the 14 cases, genomic DNA typing was successful. Analysis of mtDNA succeeded in another two cases, although genomic DNA could not be detected. Beside the accumulation of solid epidermic particles the paper describes deposition of serous and fatty tissue fluid at the ligature (mainly adjacent to skin ridges).     

Evaluation of ABO and Lewis genotypes using primer extension preamplification

There are some difficulties with blood typing from ABO variant bloodstains and Lewis negative samples using serologic methods. In these samples, DNA analysis should be employed simultaneously to avoid errors in typing. Primer extension preamplification (PEP) produces copies of template DNA. The minimum quantity to examine nucleotide substitutions of ABO and Lewis genotypes by PCR ranged from 1 to 3 ng DNA. The PCR products with or without PEP treatment showed identical ABO and Lewis genotyping results. Performing both serologic and PCR testing served to crosscheck the ABO and Lewis grouping of such specimens. Errors in ABO and Lewis typing can be avoided as discrepancies are investigated further. The application of the PEP method to limited amounts of DNA samples for ABO and Lewis blood groupings is useful.     

The primary investigation of material evidence and the evaluation of its suitability for expert biological identification by genomic "dactyloscopy"

Use of DNA "fingerprinting" method for biological analysis of material evidences exhibit new potentials for making concrete expert conclusions. But such expertise is time consuming and difficult to perform that's why it is important to evaluate fitness of expert material for DNA fingerprinting at early stages of object investigation. Method of preliminary treatment of objects sent for DNA fingerprint expertise in case of sexual assault is suggested and tested. This method allows one to evaluate objects easily from the standpoint of possibility to perform such investigation.     

Visualization of biological traces on evidence by ninhydrin

Because of possible contamination of samples with PCR inhibitors and to avoid the typing of mixed profiles the source material for forensic DNA investigations should be collected as directly and securely as possible from the evidence. This approach requires a detectability of the source material which is often not given. The procedure introduced here using selected cases enables visualization of DNA-containing materials on evidence and hence controlled analysis. For this purpose the specimen is treated with ninhydrin. A following dye reaction verifies the presence of biological material, which possibly contains DNA. An impact on subsequent STR-analysis was not observed.     

DNA typing of human remains found in damp environments

DNA typing is often used to determine identity from human remains. The environment to which the material has been exposed, however, is crucial for the success of the investigation. Damp conditions in particular can cause a rapid degradation of DNA, even in bone and teeth, and thus reduce the chances of successful typing. Here, we present the results of investigations performed on four skulls that had been lying in a damp environment for periods ranging from almost 1 year to about 45 years. In none of these cases was DNA typing successful on bone or, where present, on teeth. Where remnants of brain tissue were used, however, complete STR typing was possible in one case, partial STR typing in another, and mtDNA sequencing could be carried out in three cases. These findings suggest that brain tissue is relatively resistant to putrefaction in damp environments and, unlike bone, appears to exhibit a certain degree of protection against DNA degradation.     

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.     

ABO and Lewis typing of secretion stains on nitrocellulose membranes using a new dot-blot-ELISA technique

A new method for ABO and Lewis typing of body fluids is described. It combines the advantages of a good antigen binding to nitrocellulose membranes, the need of only very small amounts of stain material and the high sensitivity of an enzyme-linked immunosorbent assay for antigen detection. This is of special interest because conventional ABO and Lewis typing of secretion stains need relatively large stain dimensions. The method is very easy to handle, does not need any expensive equipment and gives a permanent record. Furthermore the high sensitivity offers the possibility of analyzing even sweat and urine stains without the need of concentrating these extracts.     

Utility of HLA and six erythrocyte antigen systems in excluding paternity among 500 disputed cases

Six erythrocyte antigen systems and the HLA system were evaluated to establish their practical value in 500 cases of disputed paternity. The actual results were very close to predicted values. HLA testing is expected to detect 92% and red cell testing is expected to detect 67% of men falsely accused in paternity suits. The findings of this study show that HLA detected 94% and red cell testing detected 69% of 107 men falsely accused in 500 paternity cases. In order of sensitivity, Rh, MNSs, and ABO were the most useful erythrocyte marker systems. There were six out of 107 cases in which exclusions would have been undetected if red cell typing had not been performed. Five of the six cases involved "common" HLA haplotypes.     

降解生物检材DNA分析研究新进展

在法医检案过程中,如何从降解生物检材中获得正确的DNA分型是法医学界的一个难题。本文对降解生物检材DNA分析研究取得的新进展进行综述,从检案的各个环节出发对近年来提高DNA分型成功率的方法和技术进行了详细介绍,例如新型遗传标记的开发和二代测序技术的应用等。希望为降解检材的分析提供新的思考和方法。     

Typing of mitochondrial DNA from isolated cells of the human buccal epithelium

The authors have developed a method for molecular-genetic analysis of DNA from isolated cells for the purpose of forensic medical diagnostics. The method is based on the use of the laser capture microdissection (LCM) technology in combination with typing of mitochondrial DNA. Optimization of the conditions for amplification of polymorphic mtDNA loci in preparations containing minimal amounts of the genetic material was accomplished at the initial stage of the work. To this effect, the two-round polymerase chain reaction was employed that allowed the amplified material to be accumulated in the amount sufficient for sequenation. At the next stages, the system thus obtained was tested on the cell model (using isolated cells of human buccal epithelium). It was shown that the proposed method is suitable for the analysis of specific mtDNA characteristics in a single human cell.     

Some practical notes about performing polymerase chain reaction and electrophoretic fractionation of amplificated products

Practical aspects concerning standardization of molecular-genetic expertise performing with the use of the method of DNA are considered. Examples of difficulties, which can occur at nonobservance of requirements of polymerase chain reaction and electrophoresis performing, are described; practical recommendations of their elimination are given.     

STR typing of human DNA from fly larvae fed on decomposing bodies

In homicides with entomological evidence, it may be important to prove the presumed association of fly larvae to a corpse, especially if it is in doubt whether all maggots used for entomological expertise developed and fed on it. The present study demonstrates for the first time the possibility of analyzing human microsatellite DNA present in the digestive tract of necrophagous larvae that fed on decomposed bodies with a postmortem interval up to four months. The obtained human STR profiles support the association of a maggot to a specific corpse. In addition, the identification of the host species (e.g., animal source like pig) can be achieved by analysis of the cytochrome b gene. Maggots were collected from 13 corpses after various postmortem intervals and STR typing and HVR amplifications were performed using their crop contents. In seven cases, a complete STR profile was established, in two cases, an incomplete set of alleles was obtained, and in four cases, STR typing was not successful. HVR analysis was successful in all cases except one. The time of storage of the maggots and the length of the postmortem interval up to 16 weeks appeared to have no particular influence on the quality of the results.     

Y-STR DNA amplification as biological evidence in sexually assaulted female victims with no cytological detection of spermatozoa

Identification of spermatozoa is the biological evidence most often sought in specimens from rape victims. Absence of spermatozoa usually terminates biological investigations, and the victim's testimony can be contested. We assessed the utility and reliability of PCR amplification using Y-chromosomal STR polymorphisms in specimens from female victims of sexual assault with negative cytology.One hundred and four swabs without spermatozoa detected by cytology were collected from 79 alleged sexually assaulted female victims and amplification of Y-STR and of amelogenin was performed.Overall, Y-chromosome was detected and evidenced sexual penetration in 28.8% of swabs. In the population of victims examined more than 48 h after the sexual assault, Y-STR were still evidenced in 30% of the cases. These results show that swabs should be taken from victims for Y-chromosome DNA typing even after long delays between sexual assault and medical examination.     

Optimization of the biological microchip for genotyping of the AB0 locus: correction of DNA probes

The objective of the present work was to search for methodological options facilitating the improvement and optimization of the biological microchip designed for genotyping of the AB0 locus. Testing a prototype biological microchip for genotyping of the AB0 locus using a reference set of preparations with the known group specificity has demonstrated that the choice of DNA probes by theoretical calculation of their thermodynamic parameters does not necessarily yields the desired practical result. Suffice it to say that under certain conditions some cells of the biochip appear to generate artifact hybridization signals that tend to make the results of genotyping either incorrect or difficult to interpret. This problem required the adjustment of the molecular structure of DNA probes for the optimization of their hybridization properties. As a result new DNA probes have been developed and synthesized and new variants of the prototype biochip constructed to be subjected to experimental verification.