[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.

[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.

[Identification of functionally significant mutations in NAT2 gene using biological microchips].

The product of gene NAT2 (N-acetyltransferase 2) is involved in the biotransformation system and participates in detoxication of some arylamine derivatives (in particular 2-aminofluorene, 4-aminobiphenyl and 4-naphthylamine) which are strongly mutagenic and carcinogenic. It also renders toxicological and pharmacological influence on a metabolism of medical products metabolized by the enzyme. We developed a microchip for detection of 16 functionally significant mutations coding 36 alleles of gene NAT2. Combinations of these alleles allow us to reveal more than 660 genotypes, which can be divided into four groups according acetylation phenotype: "fast" (R/R), "intermediate" (R/S), "slow" (S/S) and group with average or slow acetylating (R/S or S/S) alleles. The groups "R/S or S/S" include alleles, formed by a combination of 7 mutations (191G/A, 282C/T, 341T/C, 481C/T, 590G/A, 803A/G, 857G/A), theirs cis-trans position can be revealed by restriction analysis. In 37 of 71 DNA samples we unequivocally defined NAT2-genotypes, and other 34 samples have been characterized by more than two genotypes. 16 samples out of 34 had acetylation phenotype of group "R/S or S/S", which is characterized by the following combination of mutations: 282C/T, 341T/C, 481C/T, 590G/A and 803A/G. Thus, the developed biochip is a convenient screening method for primary detection of the majority of polymorphic replacements in gene NAT2.

[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.

[Biochips in the laboratory of A. D. Mirzabekov: 1988-2007].

The article reviews the last period of A. D. Mirzabekov's scientific career. During this time, gel-based biochips were invented, studied, and introduced to practical applications in his laboratory. This work began at the early stages of the "Human Genome" project and is continuing today, including recent development of diagnostic oligonucleotide and protein biochips. This research is discussed in the context of the worldwide development of microarray technologies.

[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.

[Identification of Mycobacterium tuberculosis strains and a simultaneous identification of their drug resistance by the hybridization method on oligonucleotide microchips]. / Opredelenie shtammov Mycobacterium tuberculosis s odnovremennym opredeleniem ikh lekarstvennoi ustoichivosti metodom gibridizatsii na oligonukleotidnykh mikrochipakh.

A method of multiplex polymerase chain reaction (PCR) with subsequent hyoridization on oligonucleotide microchips was worked out to identify the Mycobacterium tuberculosis complex and to determine simultaneously the bacterial sensitivity to 2 first-line drugs, i.e. rifampin and isoniazid. The method provides for detecting above 95% of rifampin-resistant and around 80% of isoniazid-resistant strains within 1 day.

[Fluorescence of meso-tetrakis(4-(carboxy)phenyl)porphine covalently bound to oligonucleotides d(CG)5 and d(TA)5]. / Fluorestsentsiia mezo-tetrakis(4-(karboksi)fenyl)porfina, kovalentno sviazanogo s oligonukleotidami d(CG)5 i d(TA)5.

The amino-reactive derivative of tetraphenylporphine meso-tetrakis[4-(carboxy)phenyl]porphine (TCPP) was synthesized, which is characterized by a high molar absorption coefficient (epsilon 416 = 36,500 M-1.cm-1). TCPP was covalently attached to oligonucleotides d(CG)5 [d(CG)5-TCPP] and d(TA)5 [d(TA)5-TCPP]. The spectral characteristics of these complexes were studied in 0.01 M phosphate buffer, pH 7 at 23 degrees C. UV-visible absorption spectra of these complexes have a clearly pronounced Soret band at (414 +/- 1) nm for d(CG)5-TCPP and at (412 +/- 1) nm for d(TA)5-TCPP. The fluorescence spectra of these complexes have maxima at (648 +/- 2) nm for d(CG)5-TCPP and at (658 +/- 2) nm for d(TA)5-TCPP. In this study we also determined fluorescence quantum yields q and fluorescence lifetimes tau [q = 0.099 +/- 0.011, tau = (9.0 +/- 0.3) ns for d(CG)5-TCPP and q = 0.080 +/- 0.011, tau = (8.7 +/- 0.3) ns for d(TA)5-TCPP]. A temperature rise from 5 to 50 degrees C produced only slight (within 23%) emission changes in both samples studied. Taking into account: a) high fluorescence yields (q), b) weak dependence of q on temperature, c) weak q dependence of q on the oligonucleotide type, we conclude that TCPP may be used as a sensitive fluorescence label in DNA studies.

[Preparation and primary genetic analysis of Drosophila melanogaster transformants line w'lz(b)/XXywf, containing mini-white genes, integrated in the genome during P-element-dependent transformation]. / Poluchenie i pervichnyi geneticheskii analiz transformantov Drosophila melanogaster linii w'lz(b)/XXywf, soderzhashchikh gen mini-white, integrirovannyi v genom pri P-élementzavisimoi transformatsii.

Transformation of Drosophila melanogaster using P-element-based vectors yielded 129 sublines, which carried mini-white gene copies in the different genome regions. Dependence of mini-white gene expression on the location, gene dosage, and sex of the transformed individuals was analyzed. The mutation lzb was shown to suppress mini-white gene expression, the degree of suppression depending on the location and dosage of the mini-white gene.

[Isolation of Drosophila melanogaster strain y+ snw sc wa transformed by a series of P-element mediated vectors using microinjections into early embryos]. / Poluchenie osobei Drosophila melanogaster linii y+ snw sc wa, transformirovannykh seriei vektorov na osnove P-élementa s pomoshch'iu mikroin''ektsii v rannie émbriony.

P-element-mediated transformation of Drosophila melanogaster was performed and the effectivity of transformation determined using the y+snwsc wa stock. Some new D. melanogaster stocks with sne and sn+ phenotypes were obtained as well as the stocks containing bacterial "neo" gene integrated into the genome.

[Visualization of elementary structures in polytene chromosomes of Drosophila melanogaster]. / Vizualizatsiia élementarnykh struktur v politennykh khromosomakh Drosophila melanogaster.

Electron microscopic study of total polytene chromosome preparations makes it possible to reveal elementary structures of polytene chromosomes. Elementary chromosomes are detected which contain chromomeric and interchromomeric regions. Transcription is seen in puffs and in many bands. Chromatin spreading shows that both chromomeric and interchromomeric regions of elementary chromosomes consist of nucleosomes. A ribbon-like model of polytene chromosome structure is suggested and discussed. In the ribbon-like chromosome each elementary chromosome gels conjugated only with the two adjacent sister chromosomes. Conjugation occurs only in the chromomeric regions, interchromomeric regions do not conjugate. Both chromomeric and interchromomeric regions are duplicated during polytenization which ensure proportional replication of all the bands and interbands in the course of polytenization.