RESUMEN
A panel of 106 insertion/deletion (InDel) polymorphisms and a method of their genotyping on biochips were proposed as a new approach to genetic personal identification. Short lengths and low mutation rates are basic properties of InDel markers, which thus have significant advantages over short tandem repeats (STRs) widely used in forensics. The allele frequency distributions of all known InDel polymorphisms were studied in the five largest world populations (European, East Asian, South Asian, African, and American). Markers were selected to meet the following criteria: the minor allele frequency (MAF) is higher than 0.30; the physical distance between markers is greater than 3 Mb; there are no polymorphisms, tandem repeats, and palindromes in the flanking sequences; the AT/GC ratio is close to 1. A panel of 106 polymorphisms was thus formed; the average MAF was estimated at 0.396 in the five populations. The method developed for panel genotyping included one-step multiplex PCR and subsequent hybridization on a biological microarray. The average amplicon length was 72 bp. A sample of 201 residents of Moscow and St. Petersburg was tested to determine the main characteristics of the panel: the random matching probability (MP) was 1.89x 10^(-43) and the combined probability of paternity exclusion (CPE) was 0.99999999063. The method provides an alternative to molecular genetic personal identification based on the STR length variations.
Asunto(s)
Genética de Población , Mutación INDEL , Polimorfismo Genético , Humanos , Frecuencia de los Genes , Repeticiones de MicrosatéliteRESUMEN
This paper presents a method for genotyping a panel of 60 single nucleotide polymorphisms (SNPs) using single-stage PCR followed by hybridization on a hydrogel biochip. The pool of analyzed polymorphisms consists of 41 SNPs included in the HIrisPlex-S panel, 4 SNPs of the AB0 gene (261G>Del, 297A>G, 657C>T, 681G>A), markers of the AMELX and AMELY genes, and 14 SNP markers of the Y chromosome haplogroups: B (M60), C (M130), D (CTS3946), E (M5388), G (P257), H (M2920), I (U179), J (M304), L (M185), N (M231), O (M175), Q (M1105), R (P224) and T (M272). These genetic data allow one to predict the phenotype of the desired person according to the characteristics of eye, hair, skin color, AB0 blood group, sex, and genogeographic origin in the male line. The setting protocol is simplified as much as possible to facilitate the introduction of the method into practice. The distribution of allele frequencies of the studied polymorphisms, as well as AB0 blood groups among the Slavs (N = 482), originating mainly from central Russia, was established.
Asunto(s)
Sistema del Grupo Sanguíneo ABO , Cromosomas Humanos Y , Color del Ojo , Técnicas de Genotipaje , Color del Cabello , Análisis de Secuencia por Matrices de Oligonucleótidos , Pigmentación de la Piel , Sistema del Grupo Sanguíneo ABO/genética , Cromosomas Humanos Y/genética , Color del Ojo/genética , Color del Cabello/genética , Haplotipos , Humanos , Hidrogeles , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Polimorfismo de Nucleótido Simple , Pigmentación de la Piel/genética , Población Blanca/genéticaRESUMEN
The authors report the results of the demonstrative study continuing the cycle of interactive discussions pertinent to the possibility of obtaining reliable genetic information from the analysis of burnt bone fragments. Special emphasis is placed on the worthiness of these materials for genotyping of mitochondrial DNA (mtDNA) with the use of the standard analytical methods employed for the purpose of forensic medical expertise to investigate into the length polymorphism of the amplified mtDNA fragments (PAF) by means of sequencing with fluorescent detection. The study has demonstrated that the mtDNA fragments in the state suitable for genotyping can be found only in the preparations from the bone tissue exposed to the 'mild' thermal impact after which the affected bone is virtually indistinguishable from the native one as far as the outward appearance is concerned. In the cases of a more rigorous thermal impact when the bone tissue exhibits well pronounced signs of heat destruction, it should be considered as inherently unsuitable for genotyping of mtDNA. It was shown that chromosomal DNA is inferior to mtDNA in terms of heat resistance. This finding agrees with the currently adopted view, however this advantage of mtDNA is relatively insignificant from the standpoint of genotyping efficiency.
Asunto(s)
Huesos , Quemaduras/patología , ADN Mitocondrial , Antropología Forense/métodos , Genética Forense/métodos , Huesos/lesiones , Huesos/patología , ADN Mitocondrial/análisis , ADN Mitocondrial/genética , Humanos , Biología Molecular/métodosRESUMEN
The authors overview the current state of research in the field of diagnostics and identification of the signs suggesting the presence of HIV in the materials obtained from the human corpses undergoing forensic medical expertise at different stages of their post-mortem changes. Another objective of the present work was to evaluate the risk of HIV infection for the medical personnel involved in the autopsy studies taking into consideration the significance attached in different countries to the problem of anti-infectious protection of the staff of the state institutions of forensic medical expertise. The authors discuss the possibilities and limitations of the application of the methods for HIV diagnostics, such as immunoenzymatic assays. The special attention is given to the advantages of the molecular genetic methods based on the use of the specific fragments of the viral RNA genome as the diagnostic markers. The solid methodological basis for molecular genetic diagnostics of HIV infection is provided by PCR-amplification with the detection in the real-time regime. It is supposed that this approach will make it possible not only to determine, with the high degree of accuracy and specificity, the presence of the viral genome in the biological materials but also to reduce to a minimum the probability of both false-positive and false-negative responses.
Asunto(s)
Medicina Legal/métodos , Infecciones por VIH , Infecciones por VIH/diagnóstico , Infecciones por VIH/inmunología , Humanos , Pruebas Inmunológicas/métodosRESUMEN
The objective of the present study was a demonstrative consideration of the debatable problem concerning the possibility of obtaining reliable genetic information by the investigation of burned bones. The bone fragments with the identifiable external features of different degree of ignition (i.e. in the carbonized, grey- and white-burnt states) were placed in the muffle furnace for the controlled thermal treatment. The analytical suitability of these burned bone objects for genotyping was estimated with the use of standard chromosomal STR-loci multiplex genotyping panels. The results of the study cast serious doubts as regards the possibility of genotyping of chromosomal DNA extracted from the burned bones. It was shown that the exposure of the bone tissue to a temperature of 150 degrees Celsius during 2 hours can turn it into a material absolutely unsuitable for genotyping due to the loss of all individual genotypic traits. Characteristically the burned bone objects are externally indistinguishable from the native bone. At the same time, the material with the signs of the high-temperature impact visible by the unaided eye (e.g. in the carbonized, pronounced black as well as grey and white-burnt states) is altogether unsuitable for the reliable identification of the genetic profile of chromosomal DNA.
RESUMEN
The objective of the present work was to study the phenomenon of nucleotide sequence polymorphism in alleles of the STR-loci of human chromosomal DNA and to estimate its interpopulation differences with a view to the search for the molecular-genetic markers to be used as an efficient tool for the determination of belonging of the subjects of interest to a given population. We undertook the comprehensive analysis of amplified DNA fragment sequence polymorphism (AFSP) and amplified DNA fragment length polymorphism (AFLP) with the use of the PLEX-ID-TM analytical mass-spectrometry platform (Abbott Molecular, USA). The interpopulation differences were estimated in terms of the presence or the absence of single nucleotide replacements (SNP) in the STR markers based on a few population samples. Some of the loci of interest were found to have allelic variants the occurrence of which was significantly different in individual samples. Such alleles are of importance for the further investigation since they can be regarded as potential ethno-geographical markers. Their application opens up the new promising prospects for the expert detection of the ethnic affiliation of individual subjects.
RESUMEN
The objective of the present pilot investigation was to reveal and to study polymorphism of nucleotide sequence in the alleles of STR loci of human autosomal DNA with special reference to the role of this phenomenon as a source of the differences between homonymous allelic variants. The secondary objection was to evaluate the possibility of using the data thus obtained for the enhancement of the informative value of the forensic medical genotyping of STR loci by means of identification of single nucleotide polymorphisms (SNP) for the purpose of extending their allelic spectrum. The methodological basis of the study was constituted by the comprehensive amplified fragment length polymorphism (AFLP) analysis and amplified fragment sequence polymorphisms (AFSP) analysis of DNA with the use of the PLEX-ID^TM analytical mass-spectrometry platform (Abbot Molecular, USA). The study has demonstrated that polymorphism of DNA nucleotide sequence can be regarded as the possible source of enhancement of the discriminating potential of STR markers. It means that the analysis of polymorphism of DNA nucleotide sequence for genotyping AFLP-type markers of chromosomal DNA can considerably increase the effectiveness of their application as individualizing markers for the purpose of molecular genetic expertises.
Asunto(s)
Secuencia de Bases/genética , ADN/genética , Genética Forense , Repeticiones de Microsatélite/genética , Análisis del Polimorfismo de Longitud de Fragmentos Amplificados , Genotipo , Humanos , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
The objective of the present study was the molecular-genetic authentication of the remains as an indispensable condition for the evaluation of the medical hypotheses of the cause of death in 2004 of Yasser Arafat, the former Palestinian leader and the first president of the Palestinian National Administration, the Nobel Peace Prize laureate. We carried out molecular-genetic investigations aimed at establishing the circumstances and cause of the death of Yasser Arafat including the analysis of the relevant medical documentation, the examination of the burial place at Ramallah, remains, and personal belongings stored in his Al Muqata'ah residence at Ramallah. The objective of the present molecular- genetic investigations was to confirm the authenticity of the fragments of Yasser Arafat's remains available for radio-toxicological, chemical toxicological, and other laboratory studies. The reference objects were the contact traces left on the personal belongings by their owner. The aggregate probabilistic estimate of the coincidence of genotype traits of autosomal DNA, Y-chromosomal DNA, and mtDNA was at least 99,(9)29 4% which gives evidence of the genetic identity of the objects of study. It is this value (99.999999 <...> 9999999(29) 4%) that characterizes the probability that the bone fragments provided for the laboratory studies are actually authentic remains of Yasser Arafat.
Asunto(s)
Dermatoglifia del ADN/métodos , Personajes , Genética Forense/métodos , Patologia Forense/métodos , Técnicas de Diagnóstico Molecular/métodos , Insuficiencia Multiorgánica , Entierro/métodos , Historia del Siglo XXI , Humanos , Cooperación Internacional , Masculino , Insuficiencia Multiorgánica/diagnóstico , Insuficiencia Multiorgánica/etiología , Insuficiencia Multiorgánica/historia , Polonio/análisis , Polonio/química , Polonio/toxicidadRESUMEN
The objective of the present study was to evaluate the prospects for the application of the mass-spectrometric analysis for the solution of the problems facing modern forensic-medical genetics as illustrated by the example of the new experimental multiplex approach to the typing of human DNA with the use of the complex PLEX-ID platform. The validation study involved all stages of the processing chain. The results of the study were used to develop the recommendations for the optimization of the analytical system being used. The comparative analysis of the experimental PLEX-ID technology and the traditional electrophoretic system for the analysis of polymorphism of amplified DNA fragments has demonstrated the potential advantages of the mass-spectrometric technique, such as the enhanced informative value of the forensic expert evaluation of polymorphism of STR-loci due to the possibility of identifying SNP and extension in them and therefore their allelic spectrum.
Asunto(s)
Análisis del Polimorfismo de Longitud de Fragmentos Amplificados/métodos , Antropología Forense/métodos , Genética Forense , Espectrometría de Masas/métodos , Dermatoglifia del ADN/tendencias , Genética Forense/métodos , Genética Forense/tendencias , Humanos , Reproducibilidad de los ResultadosRESUMEN
We have studied the samples of dry blood immobilized on the FTA cards following a 15-year period of their storage at room temperature. The DNA preparations were obtained based on the protocol recommended by the manufacturer including washing up with the FTA reactant and in parallel by means of complete extraction with the use of robotic stations. The preparations were compared in terms of the content of amplificationally active DNA and the effectiveness of typing STR-loci of chromosomal DNA. The complete genetic profile was derived only from 41 (82%) of the 50 FTA cards with immobilized blood samples available for the investigation that had been treated with the FTA reactant and stored during 15 years at room temperature. In 9 (18%) cases, incomplete genetic profiles were obtained that were characterized by the absence of PCR-products, as well as missing of true alleles and the presence of pseudoalleles. Such poor results are supposed to be attributable to the occurrence of the residual inhibitors of DNA-polymerase activity due to the incomplete purification of the samples. This disadvantage was overcome by the application of robotic stations for the total DNA extraction. This approach made it possible to obtain the acceptable genetic profiles for each blood samples stored at the FTA cards during 15 years. Nevertheless, even these preparations exhibited the reduced matrix activity of high molecular weight SRT-loci. This observation suggests that long-term storage of dry blood samples on FTA cards at room temperature does not guarantee the absence of degradation of their DNA.
Asunto(s)
Manchas de Sangre , Dermatoglifia del ADN/métodos , Manejo de Especímenes , Ambiente Controlado , Genética Forense/métodos , Humanos , Reproducibilidad de los Resultados , Manejo de Especímenes/métodos , Manejo de Especímenes/normas , Temperatura , TiempoRESUMEN
This study was designed to estimate the effectiveness of special technical procedures for the enhancement of sensitivity of multiplex analysis of DNA, such as the use of low-plexity PCR systems and the whole genome preamplification technology, and the possibility of their application for the purpose of forensic medical genotyping of polymorphous STR-loci of chromosomal DNA in individual cells. The authors refused to use the imitation model (equivalent DNA dilutions) for the sake of obtaining the maximally informative data and chose to work with real preparations of solitary buccal epithelial cells isolated by the laser microdissection technique. It was shown that neither the use of the low-plexity multilocus PCR systems nor the whole genome pre-amplification technology makes possible reliable genotyping of STR-loci of chromosomal DNA in individual cells. The proposed techniques allow for DNA genotyping in preparations consisting of 10 diploid cells whereas the methods for reliable genotyping of STR-loci of chromosomal DNA in individual cells remains to be developed.
Asunto(s)
Identificación Biométrica/métodos , ADN , Mucosa Bucal/patología , Análisis del Polimorfismo de Longitud de Fragmentos Amplificados/métodos , Cromosomas Humanos , ADN/análisis , ADN/aislamiento & purificación , Genética Forense , Humanos , Captura por Microdisección con Láser/métodos , Reacción en Cadena de la Polimerasa Multiplex/métodosRESUMEN
Experiments with the use of the laser capture microdissection (LCM) technology for the purpose of forensic molecular-genetic analysis carried out on real objects revealed a number of specific aspects of practical LCM application. Some of these problems have been investigated in the present work with special reference to the characteristics of the cells of interest and their physical properties in study objects. The data obtained give reason to conclude that a failure of genotyping of individual cells obtained by the LCM technique may be due to the lack of genetic material suitable for analysis. Another important cause is poor availability of the genetic material for PCR attributable to the resistance of cells subjected to the prolonged influence of environmental factors to thermal destruction. In order to obviate this difficulty, we developed a highly efficacious system with the use of PCR lysis reagents having the advantage of combination of proteolysis and the use of detergents compatible with PCR.
Asunto(s)
Contaminación de ADN , Genética Forense/métodos , Pruebas Genéticas/métodos , Captura por Microdisección con Láser/métodos , Testimonio de Experto , Humanos , Técnicas de Diagnóstico Molecular/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reproducibilidad de los ResultadosRESUMEN
The present study was designed to estimate the possibilities of application of the laser capture microdissection (LCM) technology for the molecular-genetic expert analysis (genotyping) of human chromosomal DNA. The experimental method employed for the purpose was the multiplex multilocus analysis of autosomal DNA polymorphism in the preparations of buccal epitheliocytes obtained by LCM. The key principles of the study were the application of physical methods for contrast enhancement of the micropreparations (such as phase-contrast microscopy and dark-field microscopy) and PCR-compatible cell lysis. Genotyping was carried out with the use of AmpFISTR Minifiler TM PCR Amplification Kits ("Applied Biosynthesis", USA). It was shown that the technique employed in the present study ensures reliable genotyping of human chromosomal DNA in the pooled preparations containing 10-20 dissected diploid cells each. This result fairly well agrees with the calculated sensitivity of the method. A few practical recommendations are offered.
Asunto(s)
Análisis del Polimorfismo de Longitud de Fragmentos Amplificados/métodos , Identificación Biométrica/métodos , Cromosomas Humanos/genética , ADN/genética , Genética Forense/métodos , Técnicas de Genotipaje/métodos , Captura por Microdisección con Láser/métodos , Análisis del Polimorfismo de Longitud de Fragmentos Amplificados/instrumentación , Identificación Biométrica/instrumentación , ADN/aislamiento & purificación , Células Epiteliales , Genética Forense/instrumentación , Técnicas de Genotipaje/instrumentación , Humanos , Captura por Microdisección con Láser/instrumentación , Mucosa Bucal/citologíaRESUMEN
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.
Asunto(s)
ADN Mitocondrial/genética , Genética Forense/métodos , Mucosa Bucal/citología , Humanos , Captura por Microdisección con Láser/métodos , Reacción en Cadena de la Polimerasa/métodos , Análisis de Secuencia de ADN/métodosRESUMEN
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.
Asunto(s)
Sistema del Grupo Sanguíneo ABO/genética , Tipificación y Pruebas Cruzadas Sanguíneas/métodos , Sondas de ADN/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Polimorfismo de Nucleótido Simple , Sistema del Grupo Sanguíneo ABO/sangre , Tipificación y Pruebas Cruzadas Sanguíneas/instrumentación , Sitios Genéticos , Genotipo , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos/instrumentación , Saliva/químicaRESUMEN
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.
Asunto(s)
Sistema del Grupo Sanguíneo ABO/genética , Tipificación y Pruebas Cruzadas Sanguíneas/métodos , Tipificación y Pruebas Cruzadas Sanguíneas/normas , Sondas de ADN/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/normas , Sitios Genéticos/genética , Genotipo , HumanosRESUMEN
A new approach to typing human mitochondrial DNA (mtDNA) is described using multiplex PCR in combination with genetic analysis assisted by high performance mass-spectrometry based on an automated PLEX-ID ESI-TOF analyzer (Abbot Molecular, USA). The proposed test-system is designed for DNA analysis in a standard 96-well plate and allows to simultaneously treat up to 12 samples. All stages of the analysis are fully automated, from preparation and purification of amplicons to quality control and interpretation of final results. The in-built software makes possible comprehensive analysis of the data obtained including decoding of primary results; search for, comparison and registration of polymorphism profiles in relevant databases, both directly in the nucleotide composition format and indirectly in the form of nucleotide sequences. The entire process from sample preparation to final report takes less than 8 hours to be completed. It is concluded that the PLEX-ID analytical system may be used to resolve complex problems due to the possibility to combine gene typing and individualization of the biological sample in the framework of a single analytical process starting from the sampling procedure.
Asunto(s)
Dermatoglifia del ADN/métodos , ADN Mitocondrial/genética , Genética Forense/métodos , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo Genético , Análisis de Secuencia de ADN/métodos , Dermatoglifia del ADN/tendencias , Bases de Datos Genéticas , Genética Forense/tendencias , HumanosRESUMEN
A method is proposed allowing to identify individual mitotypes in mixed preparation of mitochondrial DNA (mtDNA). Analysis of ambiguous localization on a standard electrophoregram during typing of a mixture of individual mtDNA was used to simulate conditions for selective amplification of one of the components, i.e. for its generation as a monoproduct of the polymerase chain reaction, that was followed by its sequenation and identification of individual mitotype. The possibility to use selective amplification of mtDNA sequences for differentiation between mitotypes in mixed samples is discussed with reference to its application in forensic medical examination.
Asunto(s)
ADN Mitocondrial/análisis , Genética Forense/métodos , Reacción en Cadena de la Polimerasa/métodos , Análisis de Secuencia de ADN/métodos , ADN Mitocondrial/genética , HumanosRESUMEN
The authors illustrate positive experience in organization and coordination of joint actions of expert divisions of different sectors during the accident at P.S. Podporozniy Sayano-Shushenskaya hydroelectric power station in August 2009. Special emphasis is laid on the participation of experts of quick-reaction teams formed by territorial forensic medical bureaus, mobile and supporting forces from the adjacent regions.
Asunto(s)
Accidentes de Trabajo , Medicina Legal/organización & administración , Medicina Legal/normas , Centrales Eléctricas , Femenino , Medicina Legal/métodos , Humanos , Masculino , Trabajo de Rescate/métodos , Trabajo de Rescate/organización & administración , Trabajo de Rescate/normas , SiberiaRESUMEN
This investigation was carried out in the framework of a criminal case concerning substitution of a newborn infant. The subjects involved in the case were the child and its putative mother. Solution of the problem required typing two hypervariable mitochondrial DNA markers and 82 chromosomal markers. Moreover, we had to depart from the current norms according to which parentage can be excluded if two loci of the child contain alleles absent in the reputed parent. The present examination was complicated by the manifestation of a rare genomic mutation (unipaternal disomy) that necessitated extensive studies and taking non-trivial decisions. Details of this unusual case are highlighted and difficulties encountered in the course of investigation discussed to promote future analyses of chromosomal DNA mutations in parental gametes that hamper unambiguous interpretation of results of forensic examination.