[An association study of polymorphisms in HTR2A, BDNF and SLC6A4 genes with paranoid schizophrenia and suicidal behavior].

We have developed a biochip for the analysis of candidate genes for schizophrenia. Using this biochip, allele and genotype frequencies for the polymorphisms of HTR2A, BDNF and SLC6A4 genes in 198 patients with schizophrenia and 192 healthy individuals have been obtained. The allele T of the HTR2A polymorphism rs6314 was identified as protective against the development of paranoid schizophrenia (p=0,014). An analysis of gene-gene interactions using the Multifactor-Dimensionality Reduction (MDR) algorithm has shown a statistically significant association of combined genotypes rs6311 G/-, rs6313 C/-, rs6314 C/C, rs7997012 G/- with the disease (p=0.019). Also it has been shown that the G/G genotype of the polymorphism rs6311 (p=0.013) and the C/C genotype of the polymorphism rs6313 (p=0.008) in the HTR2A gene are associated with the suicide attempt in schizophrenic patients. Correspondingly, an A allele, А/- genotypes of the polymorphism rs6311 G>A and a T allele, T/- genotypes of the polymorphism rs6313 C>T were found to be less frequent in schizophrenic patients with a history of suicide attempt than in schizophrenic patients without a history of suicide attempt, thus suggesting their protective role in the development of suicidal behavior. The results confirm the hypothesis that the HTR2A plays an important role in the etiology of schizophrenia and suicidal behavior.

[Biophysical methods for biochip analysis. Use of wide field digital fluorescent microscopy].

This paper discusses an issue on the development of biophysical methods for biochip analysis. A scheme and construction of a biochip analyzer based on wide-field digital fluorescence microscopy are described. The analyzer is designed to register images of biological microchips labeled with fluorescent dyes. The device developed is useful for high-sensitive throughput recording analyses by biochips after interaction of immobilized probes with fluorescently labeled sample molecules as well as it provides the higher rate of the analysis compared to laser scanning devices. With this analyzer a scope where biological microchips can be applied becomes wider, the development of new protocols of the analyses is possible and standard analyses run faster with the use of biochips, the expenses for the analysis performance can be reduced.

[Association study of renin-angiotensin system genes and hemostasis system genes with ischemic stroke among Russians of Central Russia].

The analysis of alleles and genotypes frequencies of 14 SNP in genes of rennin-angiotensin system (REN, AGT, AGTR1, AGTR2, BKR2, ADRB2) and hemostasis system (FGB, F2, F5, F7, ITGB3, SERPINE1, MTHFR), as well as ACE insertion-deletion polymorphism in patients with stroke comparing to healthy controls matched by age, sex and ethnicity has been carried out. The genotyping procedure included the amplification of selected gene sequences following by hybridization of fluorescently labeled fragments with SNP-specific DNA probes. The analysis of allele frequencies of each gene separately revealed no statistically significant differences between groups of patients with stroke and healthy donors. Also the complex study has been performed to estimate the contribution of rennin-angiotensin system and hemostasis system genes to the genetic susceptibility to ischemic stroke among Russians from Central Russia using method MDR (Multifactor Dimensionality Reduction). The combination with increased risk for development of ischemic stroke was presented by complex genotype FGB G/- x ACE I/- x MTHFR C/- x SERPINE1 5G/5G (p = 0.03, OR = 2.4, 95% CI 1.1-5.3), which frequency was statistically significant higher in patients with stroke compared to healthy control.

[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%.

[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.

[Microchips based on three dimensional gel cells: history and perspective]. / Mikrochipy na osnove trekhmernykh iacheek gelia: istoriia i perspektivy.

The review describes the history of creation and development of the microchip technology and its role in the human genome project in Russia. The emphasis is placed on the three-dimensional gel-based microchips developed at the Center of Biological Microchips headed by A.D. Mirzabekov since 1988. The gel-based chips of the last generation, IMAGE chips (Immobilized Micro Array of Gel Elements), have a number of advantages over the previous versions. The microchips are manufactured by photo-initiated copolymerization of gel components and immobilized molecules (DNA, proteins, and ligands). This ensures an even distribution of the immobilized probe throughout the microchip gel element with a high yield (about 50% for oligonucleotides). The use of methacrylamide as a main component of the polymerization mixture resulted in a substantial increase of gel porosity without affecting its mechanical strength and stability, which allowed one to work with the DNA fragments of up to 500 nt in length, as well as with rather large protein molecules. At present, the gel-based microchips are widely applied to address different problems. The generic microchips containing a complete set of possible hexanucleotides are used to reveal the DNA motifs binding with different proteins and to study the DNA-protein interactions. The oligonucleotide microchips are a cheap and reliable tool of diagnostics designed for mass application. Biochips have been developed for identification of the tuberculosis pathogen and its antibiotic-resistant forms; for diagnostics of orthopoxviruses, including the smallpox virus; for diagnostics of the anthrax pathogen; and for identification of chromosomal rearrangements in leukemia patients. The protein microchips can be adapted for further use in proteomics. Bacterial and yeast cells were also immobilized in the gel, maintaining their viability, which open a wide potential for creation biosensors on the basis of microchips.

[System of distributed storage and analysis of genomic information]. / Sistema raspredelennogo khraneniia i analiza genomnoi informatsii.

A distributed computing system is developed to search and analyze genetic databases using parallel computing technologies. Queries are processed by a local network PC cluster. A universal task and data exchange format is developed for effective query processing. A multilevel hierarchic task batching procedure is elaborated to generate multiple subtasks and distribute them over cluster units under dynamic priority levels and with dynamic distribution of replicated source data subbases. Primary source data preparation and generation of annotation word indices are used to significantly reduce query processing time.

[Biological microchips with hydrogel-immobilized nucleic acids, proteins, and other compounds: properties and applications in genomics]. / Biologicheskie mikrochipy, soderzhashchie immobilizovannye v gidrogele nukleinovye kisloty, belki i drugie soedineniia: svoistva i prilozheniia v genomike.

The MAGIChip (MicroArrays of Gel-Immobilized Compounds on a chip) consists of an array of hydrophilic gel pads fixed on a hydrophobic glass surface. These pads of several picoliters to several nanoliters in volume contain the gel-immobilized nucleic acids, proteins, and other compounds, as well as live cells. They are used to conduct chemical and enzymatic reactions with the immobilized compounds or samples bound to them. In the latter case, nucleic acid fragments can be hybridized, modified, and fractionated within the gel pads. The main procedures required to analyze nucleic acid sequences (PCR, detachment of primers and PCR-amplified products from a substrate, hybridization, ligation, and others) can be also performed within the microchip pads. A flexible, multipurpose, and inexpensive system has been developed to register the processes proceeding on a microchip. The system provides unique possibilities for research and biomedical applications, allowing one to register both equilibrium states and the course of reaction in real time. The system is applied to analyze both kinetic and thermodynamic characteristics of molecular interaction in the duplexes formed between nucleic acids and the probes immobilized within the microchip gel pads. Owing to the effect of stacking interaction of nucleic acids, the use of short oligonucleotides extends the possibilities of microchips for analysis of nucleic acid sequences, allowing one to employ the MALDI-TOF mass spectrometry to analyze the hybridization data. The specialized MAGIChips has been successfully applied to reveal single nucleotide polymorphism of many biologically significant genes, to identify bacteria and viruses, to detect toxins and characterize the genes of pathogenic bacteria responsible for drug resistance, and to study translocations in the human genome. On the basis of the MAGIChip, the protein microchips have been created, containing the immobilized antibodies, antigens, enzymes, and many other substances, as well as the microchips with the gel-immobilized live cells.

[Design of de novo specific DNA-binding peptides, using the motif beta-chain-turn-beta-chain for recognizing a nucleotide sequence in DNA]. / Konstruirovanie de novo spetsifichnykh DNK-sviazyvaiushchikh peptidov, ispol'zuiushchikh motiv beta-tsep'-povorot-beta-tsep' dlia uznavaniia nukleotidnoi posledovatel'nosti na DNK.

De novo design and synthesis by a solid phase technique of linear and cyclic 26-residues peptides are reported. The peptides use beta-strand-turn-beta -strand motif for sequence recognition on DNA. Amino acid sequences in the two peptides are identical, but the structure of the cyclic peptide is constrained by S-S bridge between two cysteine residues. A 28-residue peptide containing at the N-terminus a copper-chelating peptide Gly-Gly-His is also synthesized which can be used as a potential DNA-cleaving reagent. Binding of these peptides to various natural and synthetic DNAs and DNA fragment with a known base pair sequence has been studied by CD spectroscopy, fluorescence methods and DNAse I footprinting technique. By means of CD spectroscopy it is shown that 26-residue linear and cyclic peptides are partially in disordered and beta-conformations in aqueous solution in absence and in presence of 20% trifluoroethanol (TFE), but assume partially an alpha-helix conformation in the presence of 50% TFE. It is shown that linear and cyclic peptides bind to DNA. The binding approaches saturation level when one peptide molecule is bound approximately per three or four DNA base pairs. We found that antibiotic distamycin A, binding in the minor DNA groove, competes effectively with the 26-residue linear and cyclic peptides for binding to poly(dA).poly (dT). According to the CD spectroscopy data the linear and cyclic peptides undergo conformation changes upon binding to DNA, whereas the DNA structure is not markedly altered. Difference CD spectra obtained by subtracting the spectrum of the free DNA from the spectrum of the peptide-DNA mixture differ from the spectrum of the free peptide. The shapes of difference CD spectra are consistent with a conformation transition from a disordered conformation into a beta-like conformation upon binding of peptide to DNA. DNAase I footprinting diagrams show that there is a specific protection by linear and cyclic peptides of the nucleotide sequences on two ends of operators OR1, OR2 and OR3 and pseudooperators within the cro gene of 434 phage.

[Effectiveness of sequencing using stacking hybridization on oligonucleotide matrices with varying length of immobilized oligonucleotides]. / Effektivnost' provedeniia sekvenirovaniia s pomoshch'iu gibridizatsii vstyk na oligonukleotidnykh matritsakh s razlichnoi dlinoi immobilizovannykh oligonukleotidov.

The opportunity of enhancing the sequencing efficiency by applying continuous stacking hybridization is considered. The approach is based on the increase of duplex length by continuous stacking hybridization of oligonucleotides added to solution (l-oligonucleotides) to oligonucleotides immobilized on matrix (L-oligonucleotides). An analysis of reconstruction efficiency for sequenced fragments up to length of 30000 nucleotides was made. Various combinations of L- and l-oligonucleotide length were considered. The results obtained enable one to evaluate the potentialities of the proposed method for various nucleotide matrices and the complexity of experiment. Use of continuous stacking hybridization permits a considerable increase of the length of sequenced DNA fragments. We offer the approach for resolving ambiguities in branching points, which occur because of long repeats. It is based on continuous stacking hybridization of several l-oligonucleotides which form a "chain" stabilized by mutual stacking interaction.

[Measurement of distances between DNA segments for increasing the effectiveness of sequencing using hybridization on an oligonucleotide matrix]. / Izmerenie rasstoianii mezhdu uchastkami DNK dlia uvelicheniia éffektivnosti sekvenirovaniia s pomoshch'iu gibridizatsii na oligonukleotidnoi matritse.

DNA sequencing by hybridization on oligonucleotide matrix (SHOM) makes use of a matrix of immobilized oligonucleotides. Yet the method is not directly applicable for sequencing of fragments with long monotonous repeats. Measurement of the distances between certain segments within the DNA fragment analyzed provide additional information for sequencing. This information can be obtained by digesting DNA with a set of restriction endonucleases, or by PCR with primers complementary to certain DNA regions, with subsequent measurement of the length of the resulting fragments in gel electrophoresis. Use of this additional information increases the reconstruction efficiency and in many cases solves the problem of repeating and monotonous segments within analyzed DNA fragment. The current work presents the use of this information and the estimated efficiency of its usage.

[Reconstruction of a sequenced sequence using results of stacked hybridization with an oligonucleotide matrix]. / Rekonstruktsiia sekvenirumoi posledovatel'nosti po rezultatam gibridizatsii vstyk s oligonukleotidnoi matritsei.

The efficiency of additional rounds of continuous stacking hybridization in DNA sequence reconstruction by hybridization with oligonucleotide matrix (SHOM) is considered. At first, DNA is hybridized with a matrix of oligonucleotides of the length L. The overlapping of the tuples which have formed perfect duplexes with the DNA, on the one hand, has enabled us to reconstruct unambiguously a part of the sequence, and on the other hand, suggested a modified scheme for reconstructing the remaining part. Then the additional hybridizations should be carried out in the presence of shorter oligonucleotides of the length l, that are able to form perfect duplexes of the length L + l in continuous hybridization closely to matrix tuples. In this case the stability of a duplex consisting of l-tuples and long DNA is enhanced due to stacking interaction. The information obtained about the succession of L + l sites considerably increases the efficiency of reconstruction, which can eventually reach the efficiency of a matrix consisting of (L + l)-tuples. We propose here an algorithm for compiling such a set of l-tuples to be added that the number of additional hybridizations can be appreciably diminished. For an octanucleotide matrix and different sets of pentanucleotides to be used for continuous stacking hybridization, the length of unambiguously sequenced DNA undergoes a rise from 200 to some thousands bp.

[A triple-stranded "clip" from the oligonucleotide 3'-(dA)10-pO(Ch2Ch2O)3p-(dT)10-pO(CH2CH2O)3-p-(dT)10(-5)]. / Trekhtsepochechnaia "skrepka" iz oligonukleotida 3'-(dA)10-pO(CH2CH2O)3p-(dT)10-pO(CH2CH2O)3p-(dT)10(-5).

The temperature dependence of the UV- and CD-spectra of the oligonucleotides 3'-d(A)10-L-(T)10-5' [anti(AT)], 3'-d(A)10-L-d(T)10-3' [par(AT)] and 3'-d(A)10-L-(dT)10-L-(dT)10-5' [tripl(ATT)] (L = -PO(CH2CH2O) 3p-) in the phosphate buffer at pH 7 under different concentrations of NaCL and in the presence or absence of 0.01 M MgCl2 was studied. All registered structural changes are the result of intramolecular processes if the concentrations of the oligonucleotides is low (about 2.2.10(-5) M). Par(AT) and anti(AT) exist in the only two forms, transforming into each other: under low temperatures they exist as hairpins with the parallel or antiparallel orientation of chains accordingly which transform into unfolded chains when the temperature increased. In contrast trip(ATT) exists in the three different forms depending on the temperature and ion conditions. They are: the three- stranded clip, the two-stranded hairpin with a single stranded "tail" and completely unfolded chain. For the first time this work presents thermodynamic parameters of the triplex formation from deoxyoligonucleotides depending on NaCl concentration. We have registered the CD spectra to one-, two-, and three-stranded forms. Ethidium bromide binding to three-stranded "clip" was investigated, and it was established that molecules of the dye may intercalate into the "clip" with formation of stable complexes (the constant of association 10(6) M-1). It is maximum three molecules of ethidium bromid which may bound to one molecule of the three-stranded clip. It has been shown that the suggested synthetic model (three oligonucleotide blocks combined by hydroxyalkyl chains) is the most convenient for physico-chemical investigations of triplexes today.

[Parallel DNA helices. Conformational analysis of regular poly(dG).poly(dC) helices with different variants of base binding]. / Parallel'nye spirali DNK. Konformatsionnyi analiz reguliarnykh spiralei poli(dG).poli(dC) s razlichnymi variantami sviazyvaniia osnovanii.

We have performed a conformational analysis of DNA double helices with parallel directed backbone strands. The calculations were made for homopolymers poly(dG).poly(dC). All possible models of base binding were checked. By the potential energy optimization the dihedral angles and helices parameters of stable conformations of parallel double polynucleotides were calculated. The dependences of conformational energy on the base pair structure were studied. Possible structure of parallel helices with various nucleotide composition are discussed.

[Structure of d(GT)n repeating sequences with parallel and antiparallel chains]. / Struktura povtoriaiushchikhsia posledovatel'nostei d(GT)n s parallel'nymi i antiparallel'nymi tsepiami.

The ability of oligonucleotides 3'-d(GT)5pO(CH2)5Opd(GT)5-5' (anti[d(GT)]) and 3'-d(GT)5pO(CH2)6Opd(GT)5-3' (par[d(GT)]) to form hairpins and higher associates is studied. Optical methods of thermal denaturation and circular dichroism as well as the fluorescence of ethidium bromide and acridine orange bound to oligonucleotides were used. At room temperatures the formation of hairpin structure with parallel and antiparallel strands is possible. Thermodynamic parameters of par[d(GT)] and anti[d(GT)] are similar and equal to delta H = -15 kcal/mol, delta S = -50 cal/mol. deg. In the temperature range 3-10 degrees C par[d(GT)] and anti[d(GT)] form four-stranded structures with parallel chains, in which layers of four G-residues alternate with unpaired T-residues being bulged out easily. On comparison of occurrence of alternating (GT)n, (GC)n and (G)n sequences in genome it can be stated that (GT)n biological functions could be connected with conformational possibilities of the four-stranded parallel structures with unpaired T-residues.

[Interaction of trivaline with single-stranded polyribonucleotides]. / Vzaimodeistvie trivalina s odnonitevymi poliribonukleotidami.

Binding of tripeptide H-Val3-(NH)2-Dns (TVP) to polyribonucleotides was studied by fluorescence methods, circular and flow linear dichroism, equilibrium dialysis and electron microscopy. It was found that TVP binds to poly(U) in monomer, dimer and tetramer forms with binding constants of about 10(3), 40, 18.10(4) M, respectively. The cooperativity parameter for peptide dimer binding is 2000. The peptide forms tetramer complexes with poly(A), poly(C), poly(G) also. The formation of a complex between the peptide tetramer and nucleic acid is accompanied by a significant increase in the fluorescence intensity. The cooperative binding of TVP dimers to poly(U), poly(A), poly(C) is accompanied by a dramatic decrease in the flexibility of polynucleotide chains. However, it has a small effect (if any) on the flexibility of the poly(G) chain. The observed similarity of thermodynamic, optical and hydrodynamic++ properties of TVP complexes with single-stranded and double-stranded nucleic acids may reflect a similarity in the geometries of peptide complexes with nucleic acids. Electron microscopy studies show that peptide binding to poly(U) and dsDNA leads to compactization of the nucleic acids caused by interaction between the peptide tetramers bound to a nucleic acid. At the first stage of the compactization process the well-organized rod-like particles are formed, each consisting of one or more single-stranded polynucleotide fibers. Increasing the peptide concentration stimulates a side-by-side association and folding of the rods with the formation of macromolecular "leech-like" structures with the thickness of 20-50 nm.

[Hybridization of DNA with oligonucleotides immobilized in a gel: a convenient method for recording single base replacements]. / Gibridizatsiia DNK s oligonukleotidami, immobilizovannymi v gele: udobnyi metod registratsii odinochnykh zamen osnovanii.

In an attempt to develop a reliable system for DNA sequence analysis with multiple hybridization probes, oligonucleotides down to 8 bases long were covalently immobilized in a thin layer of polyacrylamide gel fixed on a glass plate. It was shown possible to detect single base changes in DNA by hybridization of the immobilized oligonucleotides with radioactively and fluorescently labeled DNA fragments. Moreover, it was found that dissociation temperatures of differently GC-rich duplexes could be equalized by appropriate choice of immobilized oligonucleotides concentrations. A model accounting for this phenomenon is presented. In order to make the system more compact, a rectangular matrix of 200 mm dots of immobilized oligonucleotides ("hybridization chip") was designed which offered the sensitivity of 20 attomoles per dot for fluorescent DNA fragment. The applications and perspectives of the approach are discussed.

[Optimal chips for megabase DNA sequencing]. / Optimal'nye chipy dlia megabaznogo sekvenirovaniia DNK.

The SHOM method (Sequencing by Hybridization with Oligonucleotide Matrix) developed in 1988 is a new approach to nucleic acid sequencing by hybridization to a octanucleotide matrix composed of an array of immobilized oligonucleotides. The original matrix proposed for sequencing by SHOM had to contain at least 65,536 octanucleotides. The present work describes a new family of matrices for sequencing, which allows one to reduce the number of synthesized oligonucleotides 5-15 times without essentially decreasing the resolving power of the method.

[Parallel double DNA spiral. A conformational analysis of regular spirals of poly(dA).poly(dT) with different variations of joining bases]. / Parallel'nye dvoinye spirali DNK, konformatsionnyi analiz reguliarnykh spiralei poli(dA).poli(dT) s razlichnymi variantami sviazyvaniia osnovanii.

We have performed a conformational analysis of DNA double helices poly(dA).poly(dT) with parallel directed backbone strands in heteronomic model frames. All possible models of base pairs and various mutual orientation of base pair and sugarphosphate backbones were checked. By the potential energy optimization the dihedral angles and helices parameters of stable conformations of parallel double polynucleotides were calculated. The dependences of conformational energy on the base pair structure were studied.

[Four-stranded DNA. Conformational analysis of regular spirals of poly(dT).poly(dA).poly(dA).poly(dT) with different base binding variations]. / Chetyrekhtsepochechnye spirali DNK. Konformatsionnyi analiz reguliarnykh spiralei poli(dT).poli(dA).poli(dA).poli(dT) s razlichnymi variantami sviazyvaniia osnovanii.

Conformational analysis of four stranded DNA helices poly(dT).poly(dA).poly(dA).poly(dT) with parallel arrangement of the identical sugar-phosphate chains connected by twofold symmetry has been performed. All possible models of symmetrical base binding were checked. By the potential energy optimization the dihedral angles and helices parameters of stable conformations of four stranded polynucleotides were calculated. The dependences of conformational energy on the base complex structure and mutual orientation of the poly(dA).and poly(dT) chains were studied. Possible biological functions of four stranded helices are discussed.