[DNA Phenotyping of Remains from Elite Burials of the Khazar Period of Southern Russia].

Ancient DNA analyses help to solve the problems related to the genogeographic origin and migration patterns of populations. The Khazar Khaganate is a subject of controversy among researchers. Its complex historical development, lack of a sufficient number of artistic and written sources, the disappearance of representatives of Khazar culture leaves open the question of the appearance of the Khazars. DNA phenotyping of bone remains from elite burials of the Khazar period of Southern Russia was carried out with respect to eye color, hair color, skin color, and AB0 blood groups. Eight out of 10 individuals had brown eyes, dark hair (to varying degrees), and a predominantly dark skin during their lifetime. Individuals from two burials had gray-blue eyes, and one individual had blond hair. The most probable AB0 blood group was identified in eight people, of which five blood group 0 (I) group, four had blood group A (II), and one had blood group B (III). The allele frequency distribution was assessed for ten population-specific autosomal markers and suggested high heterogeneity for the ethnogeographic origin of the Khazars examined. The results are evidence for ethnocultural, genetic, and phenotypic diversity of the Khazar Khaganate.

[Universal Panel of Insertion/Deletion Polymorphisms and Biochip-Based Kit ChipID106 for Genetic Personal Identification].

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.

[A Biochip for Genotyping Polymorphisms Associated with Eye, Hair, Skin Color, AB0 Blood Group, Sex, Y Chromosome Core Haplogroup, and Its Application to Study the Slavic Population].

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.

[Substrate Properties of New Fluorescently Labeled Deoxycytidine Triphosphates in Enzymatic Synthesis of DNA with Polymerases of Families A and B].

The efficiency of the incorporation of fluorescently labeled derivatives of 2'-deoxycytidine in DNA synthesized de novo has been studied using PCR with Taq and Tth polymerases of family A and Vent (exo-) and Deep Vent (exo-) polymerases of family B. Four derivatives of 5'-triphosphate-2'-deoxycytidine (dCTP) have different chemical structures of the indodicarbocyanine dye and Cy5 analogue attached to position 5 of cytosine. The kinetics of the accumulation of the PCR products and the intensity of the fluorescent signals in the hybridization analysis with immobilized DNA probes depend on the modification of the fluorescently labeled dCTP counterpart, its concentration, and the type of DNA polymerase. All labeled triphosphates showed some inhibitory effects on PCR. The best balance between the efficiency of incorporating labeled cytidine derivatives and the negative effect on the PCR kinetics has been shown in the case of Hot Taq polymerase in combination with the Cy5-dCTP analogue, which contains containing electrically neutral chro-mophore, the axis of which is a continuation of the linker between the chromophore and the pyrimidine base.

[Multiplex Genotyping of Allelic Variants of Genes Involved in Metabolizing Antileukemic Drugs].

A biochip, primer set, and genotyping protocol were developed to simultaneously address 16 single nucleotide polymorphisms in antileukemic drug metabolism genes, including TPMT, ITPA, MTHFR, SLCO1B1, SLC19A1, NR3C1, GRIA1, ASNS, MTRR, and ABCB1. The genotyping procedure included a one-round multiplex polymerase chain reaction (PCR) with simultaneous incorporation of a fluorescent label into the PCR product and subsequent hybridization on a biochip with immobilized probes. The method was used to test 65 DNA samples of leukemia patients. Fluorescence signal intensity ratios in pairs of wild-type and respective mutant sequence probes were analyzed for all polymorphic markers and demonstrated high accuracy of genotyping. The reliability of genotype determination using the biochip was confirmed by direct Sanger sequencing.

[Multiplex Assay to Evaluate the Genetic Risk of Developing Human Melanoma].

A genotyping procedure based on single-step PCR and subsequent allele-specific hybridization on a hydrogel biochip was developed to address the polymorphisms of HERC2, OCA2, SLC24A4, SLC45A2, TYR, IRF4, MC1R,MITF, PIGU, MYH7B, NCOA6, and CDK10. Amplified gene fragments were fluorescently labeled in PCR, and fluorescent signals from biochip cells were detected to evaluate how efficiently the PCR product formed a perfect duplex with an immobilized probe. The analytical characteristics of hybridization analysis were estimated for several fluorophores with different optical spectra. Cyanine dyes fluorescing in the range of Cy5 and Cy7 were synthesized for the purpose and used as 5'-tags of universal primers in single-step PCR. A Cy7 analog fluorescing in the near infrared range was found to increase the sensitivity of hybridization analysis by producing a lower background signal in the cases where target gene amplification was low.

[Detection of BCR-ABL gene mutations in chronic myeloid leukemia using biochips].

A biochip-based method was developed to identify the BCR-ABL mutations that affect the thyrosine kinase domain and determine resistance to targeted therapy with thyrosine kinase inhibitors. The method is based on RT-PCR followed by allele-specific hybridization on a biochip with immobilized oligonucleotide probes. The biochip addresses 11 mutations, which are responsible for up to 85% of imatinib resistance cases. A method to decect the clinically significant mutation T315I was designed on the basis of LNA-clamped PCR and proved highly sensitive, detecting the mutation in clinical samples with a leukemic cell content of 5% or higher. The method was validated using clinical samples from chronic myeloid leukemia (CML) patients with acquired resistance to imatinib. The results of hybridization on biochip were verified by Sanger sequencing.

[Investigation of core Y-haplogroups frequency occuring in Moscow and Saint-Petersburg citizens].

A DNA collection of 239 Moscow and 62 SPB citizens has been investigated by means of a biochip for genotyping of Y-chromosome haplogroup markers: M130 (C), M145 (DE), P257 (G), M69 (H), U179 (I), M304 (J), M185 (L), M231 (N), M175 (0), P224 (R), L146 (R1a) and M343 (R1b). Haplogroup frequency distribution in populations native to Moscow and Saint-Petersburg has been obtained. Three subsamples varying in duration of residence (one, two or three generations) were compared. Increasing of J, G, R1b frequencies may be related to immigration from Caucasia and other regions.

[Development of biochip for determination of Y-haplogroups occuring in Russian populations].

Biochip has been developed which allowed to determine the following Y-chromosome haplogroups: C, DE, G, H, I, J, L, N, O, R in a DNA sample. The following SNPs were choosen as haplogroup markers: M130, M145, P257, M69, U179, M304, M185, M231, M175, P224, correspondingly. The genotyping included two-round PCR with fluorescent label incorporation into PCR product followed by hybridization with immobilized probes on biochip. The analysis of fluorescent signal ratios in pairs of immobilized probes "wild-type probe"--"group specific probe" for each of choosen polymorphic markers showed high accuracy of Y-haplogroup genotyping using biochip. The reliability of genotyping was confirmed by direct sequencing.

[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.

[Preparing of a ssDNA target in a single-round PCR with low-melt excess primer for hybridization with microarrays].

Method of ssDNA preparing in single-round PCR for microarray application is described. The approach is exemplified on genotyping of DARC gene. It is opposed to two-round PCR that consists of separate symmethric and asymmethric stages. Implementation of reaction in single round is achieved by means of low-melt excess internal primer application. The primer do not anneal during symmethric stage but after decreasing of annealing temperature on asymmethric stage. The results indicate effective oligonucleotide microarray genotyping. The approach reduces time requirements and risk of contamination.

[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.

[HLA-DQA1, AB0 and AMEL genotyping of biological material by biochips].

A genotyping method of biological material for ABO, HLA-DQA1 and AMEL loci is described. The method is based on allele-specific SNP determination using the hydrogel biochips technology. The amplified fluorescently labeled fragments of the genes were hybridized with specific DNA probes immobilized on a biochip. The allele/genotype assignment was done according to the distribution of fluorescent signal. The minimal amount of biological material is corresponded to 100 pg of DNA. The method was proved using control samples with known genotype. Using biochips 442 DNA samples belonging to the East Slavic population group were genotyped. The allele frequencies of ABO and HLA-DQA1 loci were determined. The possibility of genotyping of biological traces, including the stubs of filter cigarettes, material from the lip of the glass was demonstrated. This method can be used for genetic testing in forensic studies. The probability that the determined genotype belongs to a concrete individual was estimated as 99.6%.

[Typing of ABO locus with biological microchip as a new level in solution of problems in forensic-medical biological expertise of material evidences].

There are cases in practice when during expertise of material evidences, discrepancies between results of typing of ABO antigens and molecular-genetic typing of DNA occur. In this work, as a radical approach to objective solution of similar conflict situations, for some contradictory case of expertise, all examinations were performed on the unified methodological base--DNA level. Instead of biological (isoserological) typing of ABO antigen, molecular-genetic typing of ABO locus with biological microchip was performed. In all cases the results, received with the use of biological microchip, do not contradict but completely conform to the results of others molecular-genetic examinations performed in the case. Given results indicate irrationality of further use of traditional methods of isoserological typing of ABO antigen for primary differentiation of biological material. These analyses, if necessary, have to be performed on DNA level with molecular-genetic expertise.

[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.

[Alginate gel microchip for real-time monitoring of intracellular processes in bacterial and yeast cells].

A method of alginate-based hydrogel cell microchip manufacturing is proposed. The development of mild conditions for cell immobilization in microvolumes of non-toxic alginate gel allows extending the range of microorganisms used. Different approaches to cell analysis using microchip have been approved in pilot studies. By the example of Escherichia coli, Bordetella bronchiseptica and Saccharomyces cerevisiae it is shown that cell microchip can be successfully applied for monitoring of nucleic acid and protein synthesis in growing cells simultaneously using two fluorescent dyes. The influence of chloramphenicol on the nucleic acids and protein synthesis in five bacterial strains has been studied on the microchip. The microchip was also applied for the analysis of inducible fluorescent protein EGFP synthesis in E. coli cells, the correlation between the level of EGFP synthesis and concentration of the inductor in the medium has been established.

Biosensing and monitoring of cell populations using the hydrogel bacterial microchip.

Advanced development of the hydrogel bacterial microchip (HBMChip) technique is proposed. The microchip represents an array of hemispherical gel elements 0.3-60 nl in volume attached to hydrophobic glass surface and containing live immobilized microbial cells. Separate gel elements contain each up to 10(5) cells and retain them inside even while the cells are dividing. Porous structure of the gel provides easy access of nutrients and tested substances to the immobilized cells. Optical signals from the cells are easily measurable and allow monitoring of intracellular metabolism using vital fluorescent stains or engineered constructs encoding bioluminescent or fluorescent reporters. Two possible application modes of the HBMChip have been investigated, i.e. the observation of bacteria and biosensing. The dynamics of nucleic acids synthesis in growing E. coli cells has been analyzed using vital fluorescent stain SYTO 9. A special function has been suggested for evaluation of the cell growth parameters. Biosensing properties of the HBMChip have been illustrated by quantitative analysis of antibiotics and the detection of sodium meta-arsenite.