PubMed · 5738195
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J C Salvidea, G E Diaz, J M Almanza. 1968-05-10. [A technic in lymphography].. https://pubmed.ncbi.nlm.nih.gov/5738195/
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BACKGROUND: Homogeneous PCR methods for genotyping usually require fluorescently labeled oligonucleotide probes. Amplicon melting with the DNA dye LCGreen I was recently introduced as a closed-tube method of genotyping that does not require probes or real-time PCR. However, some single-nucleotide polymorphisms (SNPs) could not be completely genotyped without addition of a known genotype, and high-resolution melting techniques were necessary. METHODS: A 3'-blocked, unlabeled oligonucleotide probe and the saturating dye, LCGreen I, were added to standard PCR reagents before amplification. After PCR, the samples were melted at 0.1-0.3 degrees C/s in high-resolution (HR-1), high-throughput (LightTyper), and rapid-cycle, real-time (LightCycler) instruments, and fluorescence melting curves were recorded. RESULTS: Derivative melting curves of the probe-target duplexes were characteristic of the genotype under the probe. With synthetic plasmid templates, all SNP base combinations could be genotyped. For human genomic DNA, the technique was demonstrated with mutations associated with cystic fibrosis, including SNPs (G542X, I506V, and F508C) and 3-bp deletions (F508del and I507del). CONCLUSIONS: Genotyping of SNPs and small deletions by melting analysis of an unlabeled probe in the presence of LCGreen I is simple and rapid. Only three unlabeled oligonucleotides (two primers and one probe), a saturating DNA dye, PCR, and a melting instrument are required. The method is closed-tube, does not require fluorescently labeled probes or real-time PCR, and can be completed in <10 min on any instrument capable of monitoring melting curves by fluorescence.
Commonly used dyes including Evans Blue and Trypan Blue were examined for their inhibitory activities against protein tyrosine phosphatases (PTPases), all of them showed inhibition of PTPases with different potencies. Of the 13 dyes tested, four exhibited IC(50) value of less than 10 microM, Evans Blue lowest IC(50) of 1.3 microM against PTP1B. Care must be taken in the use of dyes for clinical or biochemical experiments to avoid unwanted side effects. Some of the low molecular weight dyes might be useful as lead compounds for the development of potent and selective PTPase inhibitors.
Several methods (e.g., UV/H2O2 oxidation, adsorption, flocculation-precipitation) are normally employed to remove dye from water. A new technique based on liquid/liquid extraction using reverse micelles is proposed whereby recovery of solvent and reuse of dye is possible. Experiments were conducted by mixing a known quantity of dye in aqueous phase and solvent-containing surfactants in a simple mixer. The separation of solvent phase, containing encapsulated dye in reverse micelles, from aqueous phase due to gravity results in separation of dye from water. The removal of different ionic dyes (e.g., eosin yellow, methylene blue, malachite green, methyl orange, orange G) from aqueous phase in the presence of different cationic and anionic surfactants [e.g., sodium dodecylbenzene sulfonate, sodium bis(2-ethylhexyl) sulfosuccinate, hexadecyltrimethylammonium bromide, and cetyl pyridinium chloride] in different solvents (e.g., amyl alcohol, benzyl alcohol, methyl benzoate, and isooctane) were studied by conducting experiments. The percentage removal of dye from aqueous phase increases with the decrease in dye concentration or with the increase in surfactants concentration. Furthermore, the percentage COD removal of dye is increased with the increase in surfactant concentration. The nature of solvent has minimal effect on percentage removal of dye. The ratio of solventto aqueous phase volume required for the removal of dye decreases with the increase in surfactant concentration. It is possible to back-extract dye into aqueous phase and recover solvent by using counterionic surfactants. The separation of aqueous phase from the aqueous-phase solvent dispersion is faster for amyl alcohol as compared to benzyl alcohol and methyl benzoate. A theoretical model based on ion-exchange reaction between surfactants and dye is used to analyze the experimental data.