[The interpopulation differences between nucleotide sequence polymorphisms in alleles of the STR-loci of human chromosomal DNA]. / Mezhpopulyatsionnye razlichiya polimorfizma nukleotidnoi posledovatel'nosti v allelyakh STR-lokusov khromosomnoi DNK cheloveka.

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.

[Polymorphism of DNA nucleotide sequence as a source of enhancement of the discrimination potential of the STR-markers]. / Polimorfizm nukleotidnoi posledovatel'nosti DNK kak istochnik uvelicheniya diskriminiruyushchego potentsiala STR-markerov.

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.

[The evaluation of the prospects for the application of mass-spectrometric analysis of the amplified DNA fragments for the purpose of forensic medical expertise].

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.

[Multilocus genotyping of polymorphous STR-loci of chromosomal DNA in individual cells: technical difficulties].

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.

[The estimation of possibilities for the application of the laser capture microdissection technology for the molecular-genetic expert analysis (genotyping) of human chromosomal DNA].

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.

[Certain topical aspects of the practical application of the laser microdissection technology for the purpose of forensic molecular-genetic analysis].

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.

[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.

[On the possibility to distinguish mitotypes in mixed biological specimens based on selective amplification of mitochondrial DNA sequences].

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.

[A method for preparation of DNA suitable for medico-genetic studies from heparinized blood samples].

Heparin traditionally used as an anticoagulant for blood preparations is one of the most powerful inhibitors of the polymerase chain reaction. The molecular genetic analysis for the purpose of forensic medical expertise encounters difficulties when DNA preparations obtained from heparinized samples have to be used. In our practical work, we were faced with the necessity to use heparin-treated blood samples. It turned out impossible to eliminated heparin from DNA isolated from these samples by the known methods. In order to obviate this difficulty, we have developed a special method for obtaining DNA from heparinized blood preparations suitable for the purpose of forensic medical expertise. The new technique includes the stages of preliminary extraction and purification of the blood cell fraction.