[Recombinase Polymerase Amplification for Rapid Detection of Human Bacterial Pneumonia Pathogens].

A diagnostic system based on recombinase polymerase amplification (RPA) has been developed to identify six bacterial pathogens of human pneumonia. Species-specific primers have been designed and optimized to conduct a multiplex reaction in one common volume. Labeled primers were used for reliable discrimination of amplification products that are similar in size. Identification of the pathogen was carried out by visual analysis of an electrophoregram. The analytical sensitivity of the developed multiplex RPA was 10^(2)-10^(3) copies of DNA. The specificity of the system was determined by the absence of cross-amplification of the studied DNA samples of pneumonia pathogens for each pair of primers, as well as for the DNA of Mycobacterium tuberculosis H37rv, and amounted to 100%. The execution time of the analysis is less than an 1 h, including the electrophoretic reaction control. The test system can be used in specialized clinical laboratories for rapid analysis of samples from patients with suspected pneumonia.

[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.

Genetic classification of Aegilops columnaris Zhuk. (2n=4x=28, UcUcXcXc) chromosomes based on FISH analysis and substitution patterns in common wheat × Ae. columnaris introgressive lines.

Aegilops columnaris is a tetraploid species originated from Ae. umbellulata (2n=2x=14, UU) and a yet unknown diploid grass species. Although Ae. columnaris possesses some agronomically valuable traits, such as heat and drought tolerance and resistance to pests, it has never been used in wheat breeding because of difficulties in producing hybrids and a lack of information on the relationships between Ae. columnaris and common wheat chromosomes. In this paper, we report the development of 57 wheat - Ae. columnaris introgressive lines covering 8 of the14 chromosomes of Aegilops. Based on substitution spectra of hybrids and the results of FISH analysis of the parental Ae. columnaris line with seven DNA probes, we have developed the genetic nomenclature of the Uc and Xc chromosomes. Genetic groups and genome affinities were established for 11 of 14 chromosomes; the classification of the remaining three chromosomes remains unsolved. Each Ae. columnaris chromosome was characterized on the basis of C-banding pattern and the distribution of seven DNA sequences. Introgression processes were shown to depend on the parental wheat genotype and the level of divergence of homoeologous chromosomes. We found that lines carrying chromosome 5Xc are resistant to leaf rust; therefore, this chromosome could possess novel resistance genes that have never been utilized in wheat breeding.

[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.

[Infrared fluorescent markers for microarray DNA analysis on biological microchip].

To expand the informational capabilities of molecular genetic research, on the biological microchips, new indotricarbocyanine dyes that fluoresce in the near infrared (IR) spectral region have been synthesized. The developed IR dyes were studied using a biochip-based test system for detection of mutations in the BRCA1/BRCA2 and CHECK2 genes associated with breast cancer. The fluorescent label was introduced to the analyzed DNA during PCR using primers labeled with the synthesized IR dyes. An analyzer that allows recording and processing of images of fluorescent microarrays in the IR spectral region was designed and manufactured. It has been shown that the use of the synthesized dyes enables to conduct analysis in the IR region and improve the reliability of medical diagnostic tests due to low fluorescence intensity of sample components as well as of a biochip substrate and the reagents used for analysis.

[Preparation of recombinant serpins B3 and B4 and investigation of their specific interactions with antibodies using hydrogel-based microarrays].

The objective of this work was to obtain preparations of recombinant squamous-cell carcinoma antigens (serpins B3 and B4) and to investigate their interactions with different monoclonal antibodies using hydrogel-based microarrays (biochips). Two genetic constructs encoding full-length serpin B3 and serpin B4 molecules were created to produce recombinant SPB3 and SPB4 proteins carrying a N-terminal His6-tag. Monoclonal antibodies against serpin B3 (H3, C5, H5, H81, and G9) were also obtained. An experimental gel-based biological microchip was designed to contain gel elements that carry immobilized antibodies against SPB3, immobilized commercial monoclonal SCC107 and SCC140 antibodies against squamous-cell carcinoma antigen (SCCA), and gel elements with immobilized SPB3 or SPB4. Judging by the specificity of recombinant SPB3 and SPB4, which bind to monoclonal antibodies against SCCA and, according to the manufacturer's data, can recognize conformational epitopes of both SPB3 and SPB4, it was concluded that the obtained recombinant serpins had the correct tertiary structure. A biochip-based direct immunoassay showed that SPB4 could bind effectively only to SCC107 and SCC140 antibodies, while SPB3 interacted specifically not only with these antibodies, but also with H3 and C5 monoclonal antibodies. Using biochip-based sandwich immunoassay, a pair of monoclonal antibodies SCC107/C5 that interacted specifically with serpin B3 but did not interact with serpin B4 was identified. Thus, it has been demonstrated that serpin B3 can be selectively determined in the presence of highly homologous serpin B4 using a biochip-based assay.

[The Effect of the Chromophore Charge on the Efficiency of Incorporation of Fluorescently-labeled Nucleotides in Matrix Synthesis by Taq DNA Polymerase].

In order to study the effect of an electrical charge of the chromophore, on the efficiency of incorporation of fluorescently-labeled nucleotides into DNA during PCR, three fluorescently-labeled dUPT, one of which with electroneutral and other two with positively and negatively charged dyes (Cy5 analogs), were synthesized. It is shown that dUPT, labeled with electroneutral Cy 5 analog, is most effectively incorporated into DNA when Tag polymerase is used for PCR.

[A new immuno-PCR format for serological diagnosis of colon cancer].

Anew immuno-PCR format is described that is based on detection of membrane protein CDH17 in serum exosomes. Format application allows distinction between sera samples of healthy donors and colon cancer patients. Obtained results open a possibility of serological colon cancer diagnosis in high risk groups.

[Analysis of genetic determinants of multidrug and extensively drug-resistant Mycobacterium tuberculosis using oligonucleotide microchip].

Steadily growing resistance of the tuberculosis causative agent towards a broad spectrum of anti-tuberculosis drugs calls for rapid and reliable methods for identifying the genetic determinants responsible for this resistance. In this study, we present a biochip-based method for simultaneous identification of mutations within rpoB gene associated with rifampin resistance, mutations in katG, inhA, ahpC genes responsible for isoniazid resistance, mutations within the regions of gyrA and gyrB genes leading to fluoroquinolones resistance, and mutations in the rrs gene and the eis promoter region associated with the resistance to kanamycin, capreomycin and amikacin. The oligonucleotide microchip, as the core element of this assay, provides simultaneous identification of 99 mutations in the format "one sample--one PCR--one microchip", and it makes it possible to complete analysis of multi-drug-resistant and extensively drug-resistant tuberculosis within a single day. The tests on 63 Mycobacterium tuberculosis clinical isolates with different resistance profiles using the developed approach allows us to reveal the spectrum of drug-resistance associated mutations, and to estimate the significance of the inclusion of extra genetic loci in the determination of M. tuberculosis drug resistance.

[Sequence-specificity of protein-oligonucleotide interactions and development for protein isolation using sorptive medium with immobilized protein-specific oligonucleotides].

Sequence-specificity of binding of ribonuclease binase to oligodeoxyribonucleotides immobilized in biochip gel pads was studied. Binding constants for the complexes between binase and selected oligonucleotides were measured. Oligodeoxyribonucleotides GAGAGAG and GAGAGAGAG were found to be high-specific in interaction with binase. These oligonucleotides were used as molecular probes for immobilization in a sorptive medium for subsequent isolation and concentrating of binase from diluted water solutions. Volume capacity of the developed sorptive mediums containing immobilized oligodeoxyribonucleotides GAGAGAG and GAGAGAGAG was found to be 2.6 and 2.3 mg of binase per 1 mL of sorbate correspondingly. After the procedure of affinity chromatography and elution ofbinase from sorptive medium the protein showed the same affinity of binding to oligonucleotides as the initial sample.

[Detection of KRAS mutations in tumor cells using biochips].

Somatic mutations in the KRAS gene are important markers of some types of tumors, for example, pancreatic cancer, and may be useful in early diagnostics. A biochip has been developed which allows determining most frequent mutations in 12, 13 and 61 codons of the KRAS gene. To increase the sensitivity of the method and to make possible the analysis of minor fractions of tumor cells in clinical samples the method of blocking a wild type sequence PCR amplification by LNA-oligonucleotides has been used. The product of LNA-clamp PCR was further hybridized with oligonucleotide probes, immobilized on biochip. Biochip was tested with 42 clinical DNA samples from patients with pancreatic cancer, mostly ductal adenocarcinomas. As reference methods, the RFLP analysis and sequencing were used. The developed approach allows detecting somatic mutations in the KRAS gene if the portion of tumor cells with mutation is at least 1% of whole cell population.

[Association of NAT2 polymorphism with risks to develop psoriasis and various dermatological diseases in Moscow population].

Ruacetyltransferase 2 (NAT2) is one of key enzymes of the second phase of biotransformation that metabolize genotoxic compounds such as carcinogens and mutagens in different types of cells. There is a correlation between the decreasing activity of NAT2 gene product and the sensitivity to harmful environmental factors that increase the risk of occurrence of different multifactorial diseases, including dermatological ones like psoriasis. We developed the NAT2-biochip for 17 SNPs. The biochip was been tested on 279 clinical DNA samples from 180 patients with psoriasis and 99 healthy individuals, residents of Moscow. We found only six SNPs that were significant for European populations (282C > T, 341T > C, 481C > T, 590G > A, 803A > G and 857G > A). The analysis in psoriasis group did not show any genotype association. The increase in frequency of a slow acetylation phenotype in group of patients with type II psoriasis and in group of patients with normosthenic constitution, in comparison with control group (OR = 1.76,p = 0.177 and OR = 2.07,p = 0.050, respectively) has been revealed. The results for patients smoking one or more pack of cigarettes per day, and daily alcohol drinking in comparison with the control showed an increase in frequency for the genotype 341C/C, 481T/T, 803G/G (OR = 7.42, p = 0.008 and OR = 106.11, p = 0.003, respectively). We also found an increase of frequency of genotype 341T/T, 481C/C, 590A/-, 803A/A in patients with side reactions to medical products comparing with group of healthy donors (OR = 2.05, p = 0.099). Thus, the present data show that the certain NAT2 genotypes and some styles of life can be considered as risk factors of psoriasis development in this muscovite population.

[Biochip development for polymorphism analysis in biotransformation system genes].

Large-scale population researches, diagnostics of genetic predisposition to multifactorial diseases, screening of the polymorphic loci associated with individual sensitivity to pharmaceutical preparations, require the development of effective, exact and rapid methods of analysis for detection of many mutations simultaneously. One of the most perspective methods to solve these problems is a method of allele-specific hybridization with biochips. Taking the analysis of mutations in genes CYP1A1, CYP2D6, GSTM1, GSTT1, NAT2, CYP2C9, CYP2C19 and MTHFR as an example we showed the efficiency of using the approach for identification of individual genetic polymorphism. We believe that the biochips can be also a convenient tool in pharmacogenetics researches.

[Detection of single base polymorphism in p53 gene by ligase detection reaction and rolling circle amplification on microarrays].

We combined three modern technologies of single base polymorphism detection in human genome: ligase detection reaction, rolling circle amplification and IMAGE hydro-gel microarrays. Polymorphism in target DNA was tested by selective ligation on microarray. Product of the ligase reaction was determined in microarray gel pads by rolling circle amplification. Two different methods were compared. In first, selective ligation of short oligonucleotides immobilized on microarray was used with subsequent amplification on preformed circle probe ("common circle"). The circle probe was designed especially for human genome research. In second variant, allele-specific padlock probes that may be circularized by selective ligation were immobilized on microarray. Polymorphism of codon 72 in human p53 gene was used as a biological model. It was shown that LDR/RCA on microarray is a quantitative reaction and gives high discrimination of alleles. Principles and perspectives of selective ligation and rolling circle amplification are being discussed.

Advanced method for oligonucleotide deprotection.

A new procedure for rapid deprotection of synthetic oligodeoxynucleotides has been developed. While all known deprotection methods require purification to remove the residual protective groups (e.g. benzamide) and insoluble silicates, the new procedure based on the use of an ammonia-free reagent mixture allows one to avoid the additional purification steps. The method can be applied to deprotect the oligodeoxynucleotides synthesized by using the standard protected nucleoside phosphoramidites dG(iBu), dC(Bz)and dA(Bz).

Fabrication of microarray of gel-immobilized compounds on a chip by copolymerization.

The manufacturing of microchips containing oligonucleotides and proteins immobilized within gel pads, ranging in size from 10 x 10 to 100 x 100 microns, is described. The microchips are produced by photo- or persulfate-induced copolymerization of unsaturated derivatives of biomolecules with acrylamide-bisacrylamide mixture. Oligonucleotides containing 5'-allyl or 5'-butenediol units were synthesized using standard phosphoramidite chemistry. Acryloyl residues were attached to a protein by a two-step procedure. Photopolymerization was induced by illumination of the monomer solution containing initiator with UV light through the mask. The mask was applied directly over the monomer solution or projected through a microscope. Alternatively, copolymerization was carried out in drops of aqueous solution of monomers containing ammonium persulfate. Drops with different allyl-oligonucleotides were distributed on a glass slide, and the polymerization was induced by diffusion of N,N,N',N'-tetramethylethylenediamine (TEMED) from a hexane solution that covered the aqueous drops.

[4'-Branched nucleosides. II. Synthesis of 4'-hydroxymethyl derivatives of 2',3'-anhydronucleosides of the ribo- and lyxo-series]. / 4'-Razvetvlennye nukleozidy. II. Sintez 4'-gidroksimetil'nykh proizvodnykh purinovykh 2',3'-angidronukleozidov ribo- i likso-riada.

A general synthetic method for 4'-hydroxymethyl-2',3'-anhydronucleosides from 1,2-O-isopropylidene-4-hydroxymethyl-alpha-D-xylofuranose is described. The condensation of 1,2-di-O-acetyl-3-O-methanesulphonyl-4-benzoyloxymethyl-5-O- benzoyl-D-xylofuranose with trimethylsilyl derivatives of N6-benzoyladenine and N2-palmitoylguanine in the presence of stannic chloride resulted in the corresponding nucleosides. After their treatment with NH4OH-EtOH, corresponding 2',3'-riboanhydronucleosides were isolated. Condensation of 1,2-di-O-acetyl-3-O-benzoyl-4-benzoyloxymethyl-5-O-benzoyl-D-xy lofuranose with trimethylsilyl derivatives of purines followed by selective deacetylation led to the nucleosides with free 2'-OH group. Their 2'-O-mesylation and epoxidering closure resulted in the isolation of 2',3'-anhydrolyxonucleosides with 38-44% yields. All the compounds synthesized did not inhibit HIV-1 reproduction in human H9 and PBL cell cultures nor HSV-2 and HCMV reproduction in vero cells up to 100 microM concentrations.

[4'-Branched nucleosides. I. Synthesis of 3'-deoxy-3'-amino-4'-hydroxymethylnucleosides]. / 4'-Razvetvlennye nukleozidy. I. Sintez 3'-dezoksi-3'-amino-4'-gidroksimetilnukleozidov.

The condensation of 1, 2-di-O-acetyl-3-azido-3-deoxy-4-benzoyloxymethyl-1-5-O-benzoyl-bet a-D-ribofuranose with trimethylsilyl derivatives of N6-benzoyladenine, N4-acetylcytosine and uracil in the presence of stannic chloride led to the corresponding nucleosides. After deprotection with methanolic ammonia the azidonucleosides were reduced with PPh3 into 3'-amino-3'-deoxy-4'-hydroxymethynucleosides.