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Two series of novel zwitterionic water-soluble pentamethine cyanine dyes with geometrically balanced structures were synthesized and characterized. One series of cyanine dyes is the «zwitterionic» fluorophores, which contain positively charged trimethylammonioalkyl and negatively charged sulfonate groups. The second series of cyanine dyes contains a sulfonate group on one indolenine nucleus and a carboxyl group linked through an N- acylsulfonamide bond on the other indolenine nucleus. Dye-modified deoxyuridine triphosphates were synthesized and evaluated as reagents for nucleic acid labeling using PCR with Taq DNA-polymerase. The efficiency of the incorporation of labeled nucleotides was investigated via real-time PCR and using a «KRAS-Biochip» commercial testing system. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis remains a leading cause of morbidity and mortality worldwide. This circumstance underscores the relevance of population studies of tuberculosis for transmission dynamics control. In this study, we describe a conversion of the spoligotyping of M . tuberculosis complex isolates on a platform of custom designed hydrogel microarrays (biochips). An algorithm of automated data processing and interpretation of hybridization results using online database was proposed. In total, the 445 samples were tested. Initially, 97 samples representing multiple species of M . tuberculosis complex and nontuberculous mycobacteria were used for protocol optimization and cut-off settings. The developed assay was further evaluated on the out-group of the 348 mycobacterial samples. Results showed high concordance with the conventional membrane-based spoligotyping method. Diagnostic sensitivity and diagnostic specificity of the spoligo–biochip assay were 99.1% and 100%, respectively. The analytical sensitivity was determined to be 500 genomic equivalents of mycobacterial DNA. The high sensitivity and specificity, ease of operation procedures, and the automatic processing of measured data make the developed assay a useful tool for the rapid and accurate genotyping of M. tuberculosis . [ABSTRACT FROM AUTHOR]

• dUTP modified with tyrosine or tryptophan aromatic groups have oxidation peaks at 0.5–0.7 V. • The signals of modified dUTP were 0.2–0.3 V less positive than the oxidation signal of dGTP. • The modified dUTP were efficiently incorporated by PCR into dsDNA fragments instead of dTTP. • The PCR-generated dsDNA were detected through the oxidation of their eleсtroactive 'labels'. Deoxyuridine triphosphates (dUTP) modified with tyrosine or tryptophan aromatic groups attached through –CH=CH–CH 2 –NH–C(O)–(CH 2) n – linker at the C5 position of the pyrimidine ring and the corresponding products of polymerase chain reaction (PCR) were studied by cyclic and square wave voltammetry on carbon screen printed electrodes. A strong effect of the additional functional groups on the electrochemical activity of nucleotides was revealed. In particular, 5-aminoallyl-2′-deoxyuridine-5′-triphosphates modified with indole acetic, indole-3-propionic, indole-4-butyric, or 4-hydroxyphenylacetic acids demonstrated novel well-defined oxidation peaks at 0.5–0.7 V, similar to tryptophan or tyrosine amino acids, respectively. The oxidation potential maxima for dUTP derivatives under study were about 0.2–0.3 V less positive than the oxidation potential of dGTP (the most easily oxidizable nucleotide). Moreover, dUTP derivatives modified with tyrosine or tryptophan aromatic groups were incorporated by PCR into dsDNA fragments (amplicons) instead of dTTP. The PCR-generated dsDNA fragments with modified nucleotides were detected through the oxidation of their eleсtroactive 'labels' at micromolar concentrations, while no oxidation peaks were observed for unmodified amplicons under the same conditions. The tyrosine and tryptophan aromatic groups well complement the existing palette of electroactive tags for direct detection of nucleic acids. Compared to other electroactive 'labels', the main advantage of the developed oxidizable moieties is a good compatibility with polymerase enzymes including those used in PCR. In addition, the demonstrated signal recording procedure for modified amplicons on disposable carbon electrodes renders itself suitable for in situ analysis of biological samples for biochemical and medical applications. Image, graphical abstract [ABSTRACT FROM AUTHOR]