[Bactericidal activity of colloidal silver against grampositive and gramnegative bacteria].

It was shown that colloidal silver solution prepared in cooperation with the A. F. Ioffe Physical Technical Institute of the Russian Academy of Sciences, had significant bactericidal activity. Stable bactericidal effect on gramnegative microorganisms was observed after their 2-hour exposition in the solution of colloidal silver at a concentration of 10 ppm. Grampositive capsule-forming microorganisms were less susceptible to the colloidal silver solution: their death was observed after the 4-hour exposition in the solution.

Minor groove binder-conjugated DNA probes for quantitative DNA detection by hybridization-triggered fluorescence.

Here we describe the properties of a novel class of oligonucleotide probes capable of sensitive hybridization-triggered fluorescence. These fluorogenic probes, known commercially as MGB Eclipse probes, are characterized by having a conjugated minor groove binder (MGB) ligand at the 5'-end and a fluorophore at the 3'-end. Additionally, they have an efficient quencher moiety at the 5'-end that is useful with a wide variety of fluorescent dyes. Fluorescence of the single-stranded MGB Eclipse probe is efficiently quenched by the interaction of the terminal dye and quencher groups when not hybridized. Upon hybridization to a complementary target, the MGB molecule folds into duplex and hyper-stabilizes it, allowing the use of shorter, more specific probe sequences. The 5'-MGB-quencher group also prevents nuclease digestion by Taq DNA polymerase during PCR. Because of the hybridization-triggered fluorescence and the excellent specificity imparted by the MGB, these 5'-MGB Eclipse probes have great versatility for real-time PCR applications. The high sensitivity and specificity are illustrated using single nucleotide polymorphism detection, viral load determination, and gene expression analysis.

An improved real time PCR method for simultaneous detection of C282Y and H63D mutations in the HFE gene associated with hereditary hemochromatosis.

HFE-linked hereditary hemochromatosis (HH) is one of the most common inherited diseases among individuals of Northern European ancestry. Two sites of point mutations in the HFE gene--C282Y and H63D--are associated with greater than 90% of HH cases. We have developed a sensitive real time PCR (TaqMan) 5'-nuclease assay for single nucleotide polymorphism (SNP) detection using novel DNA chemistry, and successfully applied this method to detect these mutations. Fluorogenic PCR probes, chemically modified with a minor groove binding agent to increase duplex stability, were used in single and multiplex probe closed tube formats. The probes were tested in two commercially available thermocycling fluorimeters (the Light Cycler and the ABI Prism 7700). Comparison of the results obtained from the analysis of 43 samples showed no discrepancies between our 5' nuclease assay and the restriction length polymorphism analysis, which is routinely used in hospitals. The reported real time PCR technology is ideal for the clinical setting as it is sensitive, eliminates the labor and supply costs of post-PCR steps, reduces the risk of crossover contamination, minimizes sources of error, and can be fully automated.

The role of cyclooxygenase 2 in ulcerative colitis-associated neoplasia.

Cyclooxygenase 2 (COX-2) overexpression has been described in sporadic colonic neoplasia, but its role in ulcerative colitis (UC) neoplastic progression remains unexplored. Although the specific role of cyclooxygenase in colonic neoplasia is uncertain, its inhibition by nonsteroidal anti-inflammatory drugs decreases the risk of sporadic colonic adenocarcinoma and causes regression of adenomas in familial adenomatous polyposis. To investigate the role of COX-2 in UC-associated neoplasia, we assessed COX-2 protein and mRNA expression throughout the spectrum of UC-associated neoplastic lesions in four total colectomy specimens, using immunocytochemistry and a novel TaqMan reverse transcriptase-polymerase chain reaction assay. The findings were correlated with DNA ploidy and inflammatory activity. We found COX-2 overexpression throughout the neoplastic spectrum in UC (P: < 0.0001, R:(2)=0.53), even in diploid samples that were negative for dysplasia. Overall, neoplastic change explained 53% of the variation in COX-2 expression, whereas inflammatory activity explained only 11%. COX-2 was overexpressed in all aneuploid samples and in 38% of diploid samples (P: = 0.0074). cDNA representational difference analysis was also performed and revealed that COX-2 mRNA was an up-regulated cDNA representational difference analysis difference product. COX-2 overexpression occurs early in UC-associated neoplasia, and the increase cannot be explained by inflammatory activity alone. The data suggest that COX-2-specific inhibitors may have a chemopreventative role in UC but the possibility that they could exacerbate UC inflammatory activity needs to be tested.

3'-minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures.

DNA probes with conjugated minor groove binder (MGB) groups form extremely stable duplexes with single-stranded DNA targets, allowing shorter probes to be used for hybridization based assays. In this paper, sequence specificity of 3'-MGB probes was explored. In comparison with unmodified DNA, MGB probes had higher melting temperature (T(m)) and increased specificity, especially when a mismatch was in the MGB region of the duplex. To exploit these properties, fluorogenic MGB probes were prepared and investigated in the 5'-nuclease PCR assay (real-time PCR assay, TaqMan assay). A 12mer MGB probe had the same T(m)(65 degrees C) as a no-MGB 27mer probe. The fluorogenic MGB probes were more specific for single base mismatches and fluorescence quenching was more efficient, giving increased sensitivity. A/T rich duplexes were stabilized more than G/C rich duplexes, thereby leveling probe T(m)and simplifying design. In summary, MGB probes were more sequence specific than standard DNA probes, especially for single base mismatches at elevated hybridization temperatures.

Triplex targeting of a native gene in permeabilized intact cells: covalent modification of the gene for the chemokine receptor CCR5.

A 12 nucleotide oligodeoxyribopurine tract in the gene for the chemokine receptor CCR5 has been targeted and covalently modified in intact cells by a 12mer triplex forming oligonucleotide (TFO) bearing a reactive group. A nitrogen mustard placed on the 5'-end of the purine motif TFO modified a guanine on the DNA target with high efficiency and selectivity. A new use of a guanine analog in these TFOs significantly enhanced triplex formation and efficiency of modification, as did the use of the triplex-stabilizing intercalator coralyne. This site-directed modification of a native chromosomal gene in intact human cells under conditions where many limitations of triplex formation have been partially addressed underscores the potential of this approach for gene control via site-directed mutagenesis.

Sequence-specific targeting and covalent modification of human genomic DNA.

We compare two techniques which enable selective, nucleotide-specific covalent modification of human genomic DNA, as assayed by quantitative ligation- mediated PCR. In the first, a purine motif triplex-forming oligonucleotide with a terminally appended chlorambucil was shown to label a target guanine residue adjacent to its binding site in 80% efficiency at 0.5 microM. Efficiency was higher in the presence of the triplex-stabilizing intercalator coralyne. In the second method, an oligonucleotide targeting a site containing all four bases and bearing chlorambucil on an interior base was shown to efficiently react with a specific nucleotide in the target sequence. The targeted sequence in these cases was in the DQbeta1*0302 allele of the MHC II locus.