The assay was performed with or without addition of 10 mM DTT during the circle immobilization step as indicated on the graph. Individual RCA products were visualized using fluorescence microscopy and their number was quantified using ImageJ.
These results clearly highlight the importance of meticulously ensuring consistent reaction conditions as well as to be aware of the possibility that DTT may cause unintended side reactions altering the outcome of single molecule detecting experiments and other ultrasensitive assays. Using standard molecular biological reaction conditions, we show that DTT is able to introduce single stranded nicks in double stranded DNA.
Furthermore, DTT was able to facilitate immobilization of fluorescently labelled DNA to a functionalized microscopy slide. Although being too modest to substantially affect the results of traditional bulk assay setups, the effect of DTT was strong enough to severely affect the results of a recently developed single molecule detection REEAD setup, demonstrating a potential impact on single molecule detection protocols.
Unlike previous studies investigating thiol mediated DNA effects, the reported results were obtained using reaction conditions without any added copper. Our findings underscore the importance of carefully ensuring uniformity of reaction conditions when performing single molecule studies and of being aware of buffer additives in general and DTT in particular. In this assay, we utilized the fact that nicked DNA is separated from supercoiled and relaxed DNA when electrophoresed in the presence of the DNA intercalating dye ethidium bromide.
This is due to the fact that ethidium bromide binding introduces positive supercoiling in relaxed plasmid DNA, which results in an increased mobility. Nicked plasmid DNA retains the mobility of relaxed DNA even in the presence of ethidium bromide, since the introduced overwinding escapes via the nick. The high mobility bands represent intact plasmid DNA and the retarded bands represent nicked plasmid. National Center for Biotechnology Information , U. Journal List Sensors Basel v.
Sensors Basel. Published online May Find articles by Marie Bech Andersen. Find articles by Jonas Thomsen. Find articles by Jing Wang. Nicole Jaffrezic-Renault, Academic Editor. Author information Article notes Copyright and License information Disclaimer.
Received Mar 13; Accepted May This article has been cited by other articles in PMC. Associated Data Supplementary Materials sensorss Abstract With the novel possibilities for detecting molecules of interest with extreme sensitivity also comes the risk of encountering hitherto negligible sources of error.
Introduction In recent years, game changing technical advancements within the field of biosensors have enabled researchers to measure the activity of DNA modifying enzymes with ultra-high sensitivity and to detect even a single DNA modification event [ 1 , 2 ].
Materials and Methods 2. Nicking of Supercoiled Plasmid fmol of supercoiled pBRplasmid was incubated with 0, 0. Results and Discussion 3. Open in a separate window. Figure 1. Figure 2. Figure 3. Supplementary Materials Click here for additional data file. Author Contributions B.
Conflicts of Interest The authors declare no conflict of interest. References 1. Stougaard M. Single-molecule detection of human topoisomerase I cleavage-ligation activity. ACS Nano. Flusberg B. Direct detection of DNA methylation during single-molecule, real-time sequencing.
Getz E. A comparison between the sulfhydryl reductants tris 2-carboxyethyl phosphine and dithiothreitol for use in protein biochemistry. Netto L. Zheng S. Held K. Effects of oxygen and sulphydryl-containing compounds on irradiated transforming DNA. Part I. Actions of dithiothreitol. Solen G. It cannot go in the reverse direction kinetically speaking.
Therefore, the protein sulfhydryls stay reduced, once their disulfide bonds are broken. That is why lower concentrations of DTT are needed. However, some users report good results with higher concentrations e.
Higher concentrations are recommended for proteins with large numbers of disulfide bonds. Studies have found DTT can be used to increase the efficiency of ethanol production from biofuels. Additional reading here: Effect of the reducing agent dithiothreitol on ethanol and acetic acid production by clostridium strain P Biofuels, especially bioethanol, is already used in the transportation industry as a fuel additive. Bioethanol is produced commercially from corn and other starch-rich feedstocks.
In Brazil, biofuels are produced from sugar cane. There is an ongoing research on the production of bioethanol from other sugar crops. Dithiothreitol is used as an oxidizing agent to prevent population of mixed-disulfide species. In some instances dithiothreitol may form an adduct causing it to not cyclize since there are no free thiols remaining.
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