ABSTRACT
We have made the first apparatus for fluorescent detection, monitoring the hybridization process of fluorescently labeled DNA fragments in a polyacrylamide gel. Using this, the analysis on the thermal denaturation/reassociation process of DNA fragments in the gel was employed, for improving the performance of In-Gel Competitive Reassociation (IGCR) technique, one of genome subtraction methods. We showed that Fluorescence Resonance Energy Transfer (FRET) in the gel occurred by positioning two fluorescent dyes at 3' and 5' ends of DNA fragments. The characterization of fluorescence-labelled fragments in gel and the changes of their fluorescence intensity will be reported.
Subject(s)
DNA/analysis , Electrophoresis, Polyacrylamide Gel , Fluorescence Resonance Energy Transfer/methods , Nucleic Acid Hybridization/methods , DNA/chemistry , Fluorescent Dyes , Kinetics , Nucleic Acid Denaturation , TemperatureABSTRACT
The fluorescent detection system has been introduced into the study on denaturation/reassociation process of DNA fragments in gel, for improving In-Gel Competitive Reassociation technique, one of genome subtraction methods. The annealing behaviour of the mixture of 3'-Fluorescein-labelled and 5'-Cy5-labelled DNA fragments was analysed by Fluorescence Resonance Energy Transfer (FRET) technique from donor Fluorescein to acceptor Cy5. We showed that two fluorescent dyes labelled at 3' and 5' ends of DNA fragments caused FRET in both the solution and the gel. The characterisation of fluorescence-labelled fragments in gel and the changes of their fluorescence intensity will be reported.
Subject(s)
DNA/analysis , DNA/genetics , Fluorescence Resonance Energy Transfer/methods , Genetic Techniques , Carbocyanines , Fluorescein , Fluorescent Dyes , Gels , Nucleic Acid HybridizationABSTRACT
Erythritol is a sugar alcohol produced by Aureobasidium sp. from glucose. It is 75-80% as sweet as sucrose and is also nonhygroscopic. The aim of this study was to evaluate this sugar substitute from a cariological point of view. Erythritol was neither utilized as a substrate for the lactic acid production nor for plaque formation of mutans streptococci (serotypes a-h) and certain oral microorganisms. It was not utilized for water-insoluble glucan synthesis or cellular adherence by glucosyltransferase from Streptococcus mutans PS-14 (c) and Streptococcus sobrinus 6715 (g). Finally, a significantly lower caries score (3.1 +/- 0.5; mean +/- SEM) was observed in specific pathogen-free rats infected with S. sobrinus 6715 and fed with a diet containing 26% erythritol, as compared to control rats fed with a diet containing 26% sucrose (60.5 +/- 2.0). Also, rats provided a diet containing 56% erythritol chocolate (23.8% erythritol) and challenged with S. mutans PS-14 exhibited a significantly lower caries score (6.7 +/- 0.8) compared to the sucrose chocolate group (82.8 +/- 2.8). The main conclusion from this study is therefore that erythritol is a promising sugar substitute from a cariological point of view.
Subject(s)
Cariostatic Agents/pharmacology , Erythritol/pharmacology , Sweetening Agents/pharmacology , Actinomyces/drug effects , Actinomyces/metabolism , Actinomyces viscosus/drug effects , Actinomyces viscosus/metabolism , Animals , Bacterial Adhesion , Dental Caries/etiology , Dental Plaque/microbiology , Glucosyltransferases/metabolism , In Vitro Techniques , Lactates/biosynthesis , Lactic Acid , Lactobacillus acidophilus/drug effects , Lactobacillus acidophilus/metabolism , Lacticaseibacillus casei/drug effects , Lacticaseibacillus casei/metabolism , Rats , Rats, Sprague-Dawley , Specific Pathogen-Free Organisms , Streptococcus/drug effects , Streptococcus/metabolism , Streptococcus mutans/classification , Streptococcus mutans/drug effects , Streptococcus mutans/enzymology , Streptococcus mutans/metabolism , Streptococcus sanguis/drug effects , Streptococcus sanguis/metabolism , Streptococcus sobrinus/drug effects , Streptococcus sobrinus/metabolismABSTRACT
Erythritol is a sugar alcohol which is obtained through a cultivation of glucose and Aureobasidium sp. The sugar is about 70-80% as sweet as sucrose and is also non-hygroscopic. The effect of erythritol on cariogenicities of mutans streptococci (serotype a-h) and certain oral microorganisms was studies. Erythritol was not utilized as a substrate for the growth, lactic acid production and plaque formation of mutans streptococci (serotype a-h). It did not serve as a substrate for cellular aggregation of mutants streptococci (serotype d, g, h) and was not utilized water-insoluble glucan synthesis and cellular adherence by glucosyltransferase from S. mutans PS-14 (c) or S. sorbrinus 6715 (g). Erythritol was not also utilized for the growth and lactic acid production of certain oral microorganisms although some growth was seen with Actinomyces viscosus. SPF SD rats infected with S. sobrinus 6715 were fed a diet containing 26% erythritol or 26% sucrose for 53 days. A significantly (p less than 0.01) lower caries score (mean +/- SE; 3.1 +/- 0.5) was observed in the rat fed a diet containing erythritol than the control (60.5 +/- 2.0). The caries inhibition rate is 94.9%. Also, rats infected with S. mutans PS-14 were fed a diet containing 56% erythritol chocolate or 56% sucrose chocolate for 58 days. The mean total caries score of rats fed a diet containing 56% erythritol chocolate was 6.7 +/- 0.8, while the mean total caries score of rats fed a diet containing 56% sucrose chocolate was 82.8 +/- 2.8. The value between both groups was significant at 0.01 level, and the caries inhibition rate is 91.9%.
