E. V. Kostina

Utilization of a two-standard system in real-time PCR for quantification of gene expression in the brain.

In this study, we applied for real-time PCR the two-standard system that we had worked out previously for PCR with gel-detection of products. Genomic DNA of a known concentration was used as external standard and mRNA of the DNA-dependent RNA-polymerase II was used as internal standard. It was shown that PCR with gel-detection of products and real-time PCR provide similar results and demonstrate almost identical accuracy and repeatability when the two-standard system is used. With the help of the both methods and using the two-standard system we have confirmed the link between the genetically determined freezing reaction in mice and reduced 5-HT1A receptor mRNA level in the midbrain. We have also found that the genetically determined freezing reaction in mice is not connected with changes in Tph2 gene expression.

Universal oligonucleotide microarray for sub-typing of Influenza A virus.

A universal microchip was developed for genotyping Influenza A viruses. It contains two sets of oligonucleotide probes allowing viruses to be classified by the subtypes of hemagglutinin (H1–H13, H15, H16) and neuraminidase (N1–N9). Additional sets of probes are used to detect H1N1 swine influenza viruses. Selection of probes was done in two steps. Initially, amino acid sequences specific to each subtype were identified, and then the most specific and representative oligonucleotide probes were selected. Overall, between 19 and 24 probes were used to identify each subtype of hemagglutinin (HA) and neuraminidase (NA). Genotyping included preparation of fluorescently labeled PCR amplicons of influenza virus cDNA and their hybridization to microarrays of specific oligonucleotide probes. Out of 40 samples tested, 36 unambiguously identified HA and NA subtypes of Influenza A virus.

TaqMan probes based on oligonucleotide-hairpin minor groove binder conjugates

Hybridization of TaqMan probes derived from oligonucleotides containing fluorophores (fluorescein, FAM, or tetramethylrhodamine, (Tamra)), fluorescence quenchers (BHQ1 or BHQ2), and a conjugated hairpin binder (MGB) composed of two tripyrrolcarboxamide residues connected through an aminobutyric acid residue were proposed for discrimination of single base mismatch using the real time PCR technique. Identification of A/C mismatch was shown to be highly specific for hepatitis C virus subtypes 1a and 1b with two variants of the probe (5′-3′): Tamra-ATTGAGCGGGTTTAp-BHQ2-MGB for subtype 1a and FAMATTGAGCGGGTTGAp-BHQ1-MGB for subtype 1b. Perfect duplexes (A·T-and G·C pairs) increase fluorescence in the process of amplification, whereas imperfect duplexes (A·G-and T·C pairs) induce no fluorescence changes. This phenomenon enables simultaneous genotyping of hepatitis C virus subtypes 1a and 1b.

Microarray for diagnostics of human pathogenic influenza-a virus subtypes

An oligonucleotide microarray was developed for diagnostics of human pathogenic influenza-A virus subtypes. It contained discriminating probes for H1, H2, H3, H5, H7, and H9 subtypes of hemagglutinin and for N1, N2, and N7 subtypes of neuraminidase. An additional set of probes was used for revealing the M-gene of the influenza-A virus. The proposed microarray was tested on samples of pathogenic H5N1 avian influenza virus, pandemic H1N1 swine influenza virus, and seasonal H1N1 and H3N2 influenza viruses. The microarray can be used for the analysis both of cultivated strains and clinical specimens.

Subtyping of influenza virus A hemagglutinin with a hybridization microarray

An oligonucleotide microarray for influenza A hemagglutinin subtyping was presented. The number of probes for the determination of each subtype of hemagglutinin (H1-H13, H15, H16, pandemic flu H1N1) varied from 13 to 28. When testing the microarray using 40 type-A influenza virus isolates, the hemagglutinin subtypes were unambiguously determined for 36 specimens.

Oligonucleotide microarray for the subtyping of influenza virus a neuraminidase

A microarray for the subtyping of influenza A neuraminidase is presented. The selection of oligoprobes proceeded in two steps. The first step included the selection of peptides specific for each subtype of neuraminidase. At the second step, the oligoprobes were calculated using the found peptide structures with the subsequent additional selection of the most specific and representative probes. From 19 to 24 probes were used for the determination of each neuraminidase subtype. The microarray testing for 19 samples with the most widespread types (N1 and N2) specifies in an unequivocal definition 18 of them and only 1 isolate has not been identified.

Unexpected transformation of black hole quenchers in electrophoretic purification of the fluorescein-containing TaqMan probes

ABSTRACT The fluorescence quenchers BHQ1 and BHQ2 can be modified by trace amounts of ammonium persulfate, used for initiating gel polymerization, in electrophoretic purification of TaqMan probes using a denaturing polyacrylamide gel. The case study of BHQ1 quencher has demonstrated that a Boyland–Sims reaction proceeds in the presence of ammonium persulfate to give the corresponding sulfate. The absorption maximum of the resulting quencher shifts to the short-wavelength region relative to the absorption maximum of the initial BHQ1. The TaqMan probe containing such a quencher is less efficient as compared with the probe carrying an unmodified BHQ1. The presence of fluorescein in TaqMan probe plays decisive role in this transformation: the quencher modification proceeds at a considerably lower rate when the fluorescein is absent or replaced with a rhodamine dye (for example, R6G). It is assumed that the observed reaction can take place in two ways—both in darkness and in the reaction of the quencher in an excited state due to energy transfer from the fluorophore irradiated by light.

A compact microarray for subtyping of influenza a virus

A version of the universal oligonucleotide hybridization microchip with the size of 6 × 5 spots (4 × 4 mm) has been proposed, which operates on the principle of “one spot-one subtype.” This microchip may be the prototype of a biosensor for fixation of influenza A virus and typing of 15 subtypes of hemagglutinin and 9 subtypes of neuraminidase.

A second generation universal microarray for subtyping influenza a virus

The microchip for influenza A subtyping working on the “one spot-one subtype” principle was developed. Each spot contains a set of oligonucleotide probes specific for particular subtypes of hemagglutinin, neuraminidase and matrix protein (influenza A marker). Reliability of the proposed chip is the same as for the full-size microchip for separate hemagglutinin and neuraminidase typing which was created in our group earlier. The image was visualized by labeling the analyzed nucleic acid by either fluorescent dye or biotin with the following fixation in streptavidin-gold nanoparticles and development by silver precipitation. In the second case, the image was analyzed using an ordinary scanner that essentially simplifies influenza A subtyping.

On-microchip PCR for detection of subtypes of influenza a viruses circulating in the human population

An oligonucleotide microchip was developed for the detection of the Influenza A virus subtypes circulating in the human population, i.e., pandemic H1N1 swine influenza virus and seasonal H1N1, H2N2, H3N2, H5N1, H9N2, and H7N9 viruses. The typing of influenza viruses was performed using an on-microchip PCR. We used immobilized primer probes chosen for the neuraminidase gene, which allowed determining both neuraminidase and hemagglutinin subtypes.