DNA Bands In Gels Research Articles

Lab hands and knowing toxic substances in Uganda

How does one know a toxic substance? Perhaps paradoxically, through contact with it, as I show ethnographically from a molecular biology laboratory in Uganda, a site where people routinely handle a range of chemicals, including potentially hazardous toxicants. I analyse an everyday situation in which researchers worked with ethidium bromide, a substance regularly used to stain DNA bands in gels at this Ugandan lab but also in many comparable labs elsewhere. Ethidium bromide is one of the most fear‐inspiring chemicals at this lab; it is considered a potent mutagen and thus counts as highly carcinogenic. While the lab has clear safety procedures concerning how to handle this substance correctly, these are often not implemented in a hard‐to‐control lab setting marked by both material deprivation and a steady flow of unremunerated volunteers who seek to gain practical experience. I show how experienced Ugandan scientists have learned to mitigate their own risk of exposure to ethidium bromide by developing choreographed routines that prevent them from inadvertently touching it. I examine this as a way of ‘handling’ a dangerous substance, developing and habituating skilled movements of hands and bodies that then are passed on to other researchers. It is not an abstract knowledge of a substance's chemical properties that counts here; instead, knowing a toxic substance implies practical routines of handling.

Factors affecting quantitation of DNA bands in gels using a charge-coupled device imaging system.

Quantitation of DNA bands separated in polyacrylamide or agarose gels was tested under a variety of conditions to examine key factors contributing to the ability to obtain quantitative data. Variations tested included comparison of different fluorescent stains (ethidium bromide and GelStar stain), and variations of other parameters relating to the electrophoretic separation, e.g. gel and sample geometry, mode of staining, and mode of excitation of stains. The results showed that linear results were seen for a similar 20-30-fold range of DNA concentrations with both stains tested and that it is critical to standardize separations to obtain consistent results. Variations in separation and detection could lead to relatively large changes in fluorescent signals seen for similar amounts of DNA.

Caffeine dissociates complexes between DNA and intercalating dyes: Application for bleaching fluorochrome‐stained cells for their subsequent restaining and analysis by laser scanning cytometry

Background Removal of the nucleic acid-bound fluorochrome is desirable when stained cells have to be reanalyzed using other fluorochromes. It is also often desirable to remove DNA-bound antitumor drugs from drug-treated cells, to improve cell staining. We have previously observed that in aqueous solutions, the methylxanthine caffeine (CFN) decreases interactions between planar aromatic molecules such as intercalating dyes or antitumor drugs and nucleic acids. The aim of this study was to explore whether this property of CFN can be utilized to remove the DNA-bound intercalating dyes propidium iodide (PI) or 7-aminoactinomycin D (7-AAD) from the cells and whether the bleached cells can be restained and reanalyzed. Methods HL-60 cells were fixed in 70% ethanol and their DNA was stained with PI or 7-AAD. The cells were then rinsed with a 0.05 M solution of CFN in phosphate-buffered saline (PBS) or with PBS alone. The decrease in intensity of cell fluorescence during rinsing was measured by laser scanning cytometry (LSC) to obtain the bleaching kinetics of individual cells. The bleached cells were then restained with PI, 7-AAD, or the protein-specific fluorochrome sulforhodamine 101(S101). Their fluorescence was measured again by LSC. In addition, free DNA was subjected to gel electrophoresis, DNA bands in the gels were stained with ethidium bromide (EB), and the gels were rinsed with a solution of CFN or PBS to bleach the DNA band's fluorescence. Results Rinsing the PI or 7-AAD–stained cells with solutions of CFN led to nearly complete removal of PI and a more than 75% decrease in 7-AAD fluorescence after 10 min. The rinse with PBS decreased the PI cell fluorescence intensity by less than 30% and the 7-AAD fluorescence by about 50%. The differences in kinetics of PI or 7-AAD removal by CFN from G2/M versus G1 cells suggest that these intercalators bind more strongly to DNA in chromatin of G2/M than G1 cells. The CFN-bleached cells were then successfully stained with S101 and again with PI or 7-AAD. The bivariate analysis of the LSC merged files of the cells sequentially stained with PI and S101 revealed typical DNA/protein distributions. The fluorescence of EB-stained DNA bands in gels was also nearly completely removed by rinsing gels in 0.05 M CFN; PBS alone had a distinctly lesser effect. Conclusion Solutions of CFN can dissociate the DNA-bound PI, 7-AAD, EB, and possibly other intercalating fluorochromes. The bleached cells can be restained and reanalyzed by LSC. This approach can also be used to remove such fluorochromes from nucleic acids immobilized in gels and perhaps in other solid matrices. Analysis of the kinetics of fluorochrome removal from cells can possibly be used to study their binding affinities to nucleic acids in situ. Cytometry 43:38–45, 2001. © 2001 Wiley-Liss, Inc.

Caffeine dissociates complexes between DNA and intercalating dyes: Application for bleaching fluorochrome-stained cells for their subsequent restaining and analysis by laser scanning cytometry

Removal of the nucleic acid-bound fluorochrome is desirable when stained cells have to be reanalyzed using other fluorochromes. It is also often desirable to remove DNA-bound antitumor drugs from drug-treated cells, to improve cell staining. We have previously observed that in aqueous solutions, the methylxanthine caffeine (CFN) decreases interactions between planar aromatic molecules such as intercalating dyes or antitumor drugs and nucleic acids. The aim of this study was to explore whether this property of CFN can be utilized to remove the DNA-bound intercalating dyes propidium iodide (PI) or 7-aminoactinomycin D (7-AAD) from the cells and whether the bleached cells can be restained and reanalyzed. HL-60 cells were fixed in 70% ethanol and their DNA was stained with PI or 7-AAD. The cells were then rinsed with a 0.05 M solution of CFN in phosphate-buffered saline (PBS) or with PBS alone. The decrease in intensity of cell fluorescence during rinsing was measured by laser scanning cytometry (LSC) to obtain the bleaching kinetics of individual cells. The bleached cells were then restained with PI, 7-AAD, or the protein-specific fluorochrome sulforhodamine 101(S101). Their fluorescence was measured again by LSC. In addition, free DNA was subjected to gel electrophoresis, DNA bands in the gels were stained with ethidium bromide (EB), and the gels were rinsed with a solution of CFN or PBS to bleach the DNA band's fluorescence. Rinsing the PI or 7-AAD-stained cells with solutions of CFN led to nearly complete removal of PI and a more than 75% decrease in 7-AAD fluorescence after 10 min. The rinse with PBS decreased the PI cell fluorescence intensity by less than 30% and the 7-AAD fluorescence by about 50%. The differences in kinetics of PI or 7-AAD removal by CFN from G2/M versus G1 cells suggest that these intercalators bind more strongly to DNA in chromatin of G2/M than G1 cells. The CFN-bleached cells were then successfully stained with S101 and again with PI or 7-AAD. The bivariate analysis of the LSC merged files of the cells sequentially stained with PI and S101 revealed typical DNA/protein distributions. The fluorescence of EB-stained DNA bands in gels was also nearly completely removed by rinsing gels in 0.05 M CFN; PBS alone had a distinctly lesser effect. Solutions of CFN can dissociate the DNA-bound PI, 7-AAD, EB, and possibly other intercalating fluorochromes. The bleached cells can be restained and reanalyzed by LSC. This approach can also be used to remove such fluorochromes from nucleic acids immobilized in gels and perhaps in other solid matrices. Analysis of the kinetics of fluorochrome removal from cells can possibly be used to study their binding affinities to nucleic acids in situ.