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Aims The aim of this study was to develop a rapid PCR-based method for spoligotyping of Mycobacteria in the microarray format and to compare it to conventional spoligotyping by hybridization. Methods and results The method employs the on-Chip PCR technique with primers specific for 43 spacers that separate direct repeats (DRs) in the DR region of mycobacterial DNA. The primers were immobilized on gel-based microarrays, and PCR was performed directly on the chips. The PCR fluorescence images were acquired and processed using a portable fluorescence analyzer equipped with dedicated software. Analysis takes 1.5–2 hours and can be carried out on clinical samples without additional handling. The analytical sensitivity of the method was 103 copies of target DNA. The spoligotyping results of 51 samples produced by the proposed method and by conventional reverse hybridization approach were in full concordance. Conclusions High throughput capacity, computerized data analysis, compact equipment, and reliable results make the on-Chip PCR an attractive alternative to intra- and interspecific spoligotyping of Mycobacterium tuberculosis complex bacteria. Significance and Impact of Study Fast microarray-based spoligotyping technique using on-Chip PCR was developed. [ABSTRACT FROM AUTHOR]

Two novel electroneutral indotricarbocyanine dyes with two sulfo groups have been synthesized. They possess higher photo and thermal stability as compared to known analogue and were conjugated with aminoallyl-dUTP for enzymatic labeling of DNA by polymerase chain reaction. [ABSTRACT FROM AUTHOR]

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]

[Display omitted] • Novel water-soluble cyanine dyes with terminal alkyne group were synthesized. • Highly efficient approach of the single-step Sonogashira-type cross-coupling reaction for the synthesis of 2′-deoxycytidine-5′-triphosphates bearing a novel cyanine dye attached via an acetylene linker using freeze–pumpthaw degassing methodology was developed. • Dye-modified 2′-deoxycytidine-5′-triphosphates containing an alkynyl linker with a carboxamide fragment is the most efficient substrate for Taq and Vent (exo-) DNA polymerases. New applications of palladium-catalyzed Sonogashira-type cross-coupling reaction between C5-halogenated 2′-deoxycytidine-5′-monophosphate and novel cyanine dyes with a terminal alkyne group have been developed. The present methodology allows to synthesize of fluorescently labeled C5-nucleoside triphosphates with different acetylene linkers between the fluorophore and pyrimidine base in good to excellent yields under mild reaction conditions. Modified 2′-deoxycytidine-5′-triphosphates were shown to be good substrates for DNA polymerases and were incorporated into the DNA by polymerase chain reaction. [ABSTRACT FROM AUTHOR]

The novel C5-modified nucleotides containing indodicarbocyanine dye bearing a carboxyl group at the 3-position of indolenine have been synthesized and incorporated into DNA by commercially available thermostable Taq DNA polymerase. The efficiency of incorporation of labeled nucleotides during polymerase chain reaction was studied in a TB-biochip and THROMBO-Biochip test systems. [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]