This work was supported by startup funds to TDG from Christopher Newport University.&#160;

1. Preparing the gel
NOTE: For general gel electrophoresis protocols, see also P.Y. Lee, et al.14.&#160;
<ol>
	<li>Mix agarose (~1% w/v, percentage can be varied for particular size separations) in buffer (approximately 70 mL for a mini-gel (8 x&#160;7 cm)). Buffers are commonly TAE (tris-acetate-EDTA, 40 mM Tris, 20 mM acetate, 1 mM EDTA, pH approximately 8.6) or TBE (tris-borate-EDTA, 90 mM Tris, 90 mM borate, 2 mM EDTA, pH approximately 8.3)).</li>
	<li>Add thiazole orange to a final concentration of 1.3 &#956;g/mL.
	<ol>
		<li>Dissolve thiazole orange in DMSO to make a 10,000x stock solution (13 mg/mL). While not particularly light sensitive, store TO in the dark when not in use. This solution is stable at room temperature for ~months; long-term storage may be achieved by freezing aliquots (DMSO will freeze in a standard refrigerator). Be sure to entirely melt and resuspend a frozen solution before use.&#160; 
		NOTE: The gel can also be stained with TO after electrophoresis (see step 2.6). While TO has an improved safety profile over ethidium bromide, standard molecular biology laboratory safety precautions should be maintained.&#160;</li>
	</ol>
	</li>
	<li>Microwave the mixture of agarose, buffer, and thiazole orange to dissolve agarose (approximately 60 s). This step is commonly referred to as &#8220;melting&#8221;. Swirl (5 s) to aid dissolution if needed.&#160; 
	NOTE: TO can also be added after microwaving if preferred.</li>
	<li>Allow the agarose solution to cool briefly before pouring into gel casting apparatus containing an appropriate comb.</li>
	<li>Allow the agarose solution to solidify into a gel.&#160;</li>
</ol>
2. Loading and running the gel
<ol>
	<li>Place the gel in the electrophoresis apparatus if not already present.</li>
	<li>Add running buffer (TAE or TBE as above) to cover the surface of the gel.</li>
	<li>Load DNA samples (commonly 10 &#956;L) using a loading dye. Include a DNA sizing ladder for reference.</li>
	<li>Attach the cover and electrodes (the gel should be run toward the red anode (positive)).</li>
	<li>Apply voltage (typically ~100V for a mini-gel, although size of gel may require a modified voltage to prevent damaging the gel) until loading dye has traveled an appropriate distance (approximately 4-7 cm for a mini-gel, although distance may vary depending on precise application). 
	NOTE: Like ethidium bromide and many other DNA-binding dyes, thiazole orange is positively charged. Consequently, these dyes will migrate in the opposite direction of electrophoresing DNA. For samples which are run far down the gel for enhanced separation, eventually the dye will separate from the smaller DNA fragments, resulting in weak staining. In these instances, the gel should be stained as in step 2.6. This situation is not unique to TO, any positively charged dye that reversibly interacts with DNA will exhibit this behavior (including ethidium bromide, TO, and others).</li>
	<li>If TO was not added prior to gel casting (step 1.2), stain by immersing the gel in the buffer containing thiazole orange.
	<ol>
		<li>Prepare enough buffer (TAE or TBE) containing 1.3 &#956;g/mL thiazole orange to completely cover the gel and soak the gel with gentle agitation until the bands are fully detected (roughly 20 min).</li>
	</ol>
	</li>
</ol>
3. Visualization of thiazole orange agarose gel (UV transilluminator)
<ol>
	<li>Remove the gel from the electrophoresis apparatus and place on a UV transilluminator. 
	CAUTION: UV light is damaging to skin and eyes. Be sure to wear appropriate eye (goggles) and face (face shield) protection. Hands should have gloves and long sleeves should be worn.</li>
	<li>If desired, cut out desired DNA bands from the gel (for further digestion or ligation, for example). Expose the gel to UV light for as short a length of time as possible during excision. UV light (regardless of DNA dye) damages DNA.&#160;</li>
	<li>Extract DNA from the gel slice using a readily available kit or protocol15.</li>
</ol>
4. Visualization of thiazole orange agarose gel (blue-light transilluminator or flashlight)
<ol>
	<li>Remove the gel from the electrophoresis apparatus and place on a blue-light transilluminator (~470 nm maximum emission wavelength). Alternatively, a blue LED (~470 nm) flashlight can be directed at the gel (either from above or below). 
	NOTE: While sensitivity is lower using a blue-light transilluminator with ethidium bromide, DNA stained with ethidium bromide can be detected using this blue-light protocol.</li>
	<li>Use an amber emission filter (~560 nm longpass, either goggles or square) to filter blue light, enabling visualization of fluorescence from DNA:thiazole orange complexes.&#160; 
	NOTE: Without the amber emission filter, it is very difficult to detect DNA bands due to intensity of blue-light excitation source.&#160; 
	CAUTION: Although blue light lacks the ability to acutely damage tissues (contrasting UV), prolonged exposure to intense blue light could damage eyes and amber emission goggles or filter should be used.</li>
	<li>If desired, cut out desired DNA bands from the gel for further applications. 
	NOTE: Since blue light does not damage DNA, it is not necessary to rapidly cut out bands (such as when using UV excitation in step 3.2).</li>
	<li>Extract DNA from the gel slice using a readily available kit or protocol15.</li>
</ol>
5. Image capture
<ol>
	<li>Select appropriate excitation and emission settings in gel-imaging apparatus. The excitation and emission of thiazole orange (&#955;ex,max = 510 nm (488 nm and 470 nm also show strong excitation, in addition to strong excitation at UV wavelengths); &#955;em = 527 nm) are nearly identical to common blue-light&#8211;detectable commercial dyes, so instruments may have preset filter settings that can be used.&#160; 
	NOTE: Some filter settings for blue-light&#8211;detectable commercial dyes actually use damaging UV light for excitation, so use caution if imaging prior to cutting out bands. Use a blue-light excitation source if possible when DNA bands will be excised after imaging.</li>
	<li>In the absence of an imaging system with appropriate filters, place an amber filter between the camera and the gel/blue-light excitation source.</li>
</ol>

<ol>
	<li>O'Neil, C. S., Beach, J. L., Gruber, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thiazole+orange+as+an+everyday+replacement+for+ethidium+bromide+and+costly+DNA+dyes+for+electrophoresis.">Thiazole orange as an everyday replacement for ethidium bromide and costly DNA dyes for electrophoresis.</a> Electrophoresis. 39 (12), 1474-1477 (2018).</li><li>McCann, J., Choi, E., Yamasaki, E., Ames, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+carcinogens+as+mutagens+in+the+Salmonella/microsome+test:+assay+of+300+chemicals.">Detection of carcinogens as mutagens in the Salmonella/microsome test: assay of 300 chemicals.</a> Proceedings of the National Academy of Sciences of the United States of America. 72 (12), 5135-5139 (1975).</li><li>Evenson, W. E., Boden, L. M., Muzikar, K. A., O&#8217;Leary, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=1H+and+13C+NMR+Assignments+for+the+Cyanine+Dyes+SYBR+Safe+and+Thiazole+Orange.">1H and 13C NMR Assignments for the Cyanine Dyes SYBR Safe and Thiazole Orange.</a> The Journal of Organic Chemistry. 77 (23), 10967-10971 (2012).</li><li>Beaudet, M., Cox, G., Yue, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Probes, Inc. , (2005).</li><li>Pfeifer, G. P., You, Y. H., Besaratinia, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+induced+by+ultraviolet+light.">Mutations induced by ultraviolet light.</a> Mutation Research. 571 (1-2), 19-31 (2005).</li><li>Cariello, N. F., Keohavong, P., Sanderson, B. J., Thilly, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+damage+produced+by+ethidium+bromide+staining+and+exposure+to+ultraviolet+light.">DNA damage produced by ethidium bromide staining and exposure to ultraviolet light.</a> Nucleic Acids Research. 16 (9), 4157 (1988).</li><li>Hartman, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transillumination+can+profoundly+reduce+transformation+frequencies.">Transillumination can profoundly reduce transformation frequencies.</a> BioTechniques. 11 (6), 747-748 (1991).</li><li>Lee, L. G., Chen, C. H., Chiu, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thiazole+orange:+a+new+dye+for+reticulocyte+analysis.">Thiazole orange: a new dye for reticulocyte analysis.</a> Cytometry. 7 (6), 508-517 (1986).</li><li>Nygren, J., Svanvik, N., Kubista, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Interactions+Between+the+Fluorescent+Dye+Thiazole+Orange+and+DNA.">The Interactions Between the Fluorescent Dye Thiazole Orange and DNA.</a> Biopolymers. , 1-13 (1998).</li><li>Svanvik, N., Westman, G., Wang, D., Kubista, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-Up+Probes:+Thiazole+Orange-Conjugated+Peptide+Nucleic+Acid+for+Detection+of+Target+Nucleic+Acid+in+Homogeneous+Solution.">Light-Up Probes: Thiazole Orange-Conjugated Peptide Nucleic Acid for Detection of Target Nucleic Acid in Homogeneous Solution.</a> Analytical Biochemistry. 281 (1), 26-35 (2000).</li><li>Yang, P., De Cian, A., Teulade-Fichou, M. P., Mergny, J. L., Monchaud, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+Bisquinolinium/Thiazole+Orange+Conjugates+for+Fluorescent+Sensing+of+G-Quadruplex+DNA.">Engineering Bisquinolinium/Thiazole Orange Conjugates for Fluorescent Sensing of G-Quadruplex DNA.</a> Angewandte Chemie International Edition. 48 (12), 2188-2191 (2009).</li><li>Fang, G. M., Chamiolo, J., Kankowski, S., Hovelmann, F., Friedrich, D., Lower, A., Meier, J. C., Seitz, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bright+FIT-PNA+hybridization+probe+for+the+hybridization+state+specific+analysis+of+a+C+&#8594;+U+RNA+edit+via+FRET+in+a+binary+system.">A bright FIT-PNA hybridization probe for the hybridization state specific analysis of a C &#8594; U RNA edit via FRET in a binary system.</a> Chemical Science. 9 (21), 4794-4800 (2018).</li><li>Pei, R., Rothman, J., Xie, Y., Stojanovic, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-up+properties+of+complexes+between+thiazole+orange-small+molecule+conjugates+and+aptamers.">Light-up properties of complexes between thiazole orange-small molecule conjugates and aptamers.</a> Nucleic Acids Research. 37 (8), e59-e59 (2009).</li><li>Lee, P. Y., Costumbrado, J., Hsu, C. Y., Kim, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agarose+Gel+Electrophoresis+for+the+Separation+of+DNA+Fragments.">Agarose Gel Electrophoresis for the Separation of DNA Fragments.</a> Journal of Visualized Experiments. (62), 1-5 (2012).</li><li>Vogelstein, B., Gillespie, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparative+and+analytical+purification+of+DNA+from+agarose.">Preparative and analytical purification of DNA from agarose.</a> Proceedings of the National Academy of Sciences of the United States of America. 76 (2), 615-619 (1979).</li></ol>

The authors have nothing to disclose.

Ethidium bromide has long been a standard tool in the molecular biology lab, despite known toxicity. It also suffers from requiring UV light, which damages the DNA as it is being detected. Thiazole orange offers an inexpensive alternative to ethidium bromide, as well as useful but expensive commercial dyes.&#160;
The benefits of thiazole orange are thus two-fold. First, thiazole orange can simply be used as a replacement to ethidium bromide. Gels can be prepared identically to EtBr, with TO su...

The purpose of this method is to identify DNA in agarose gels using thiazole orange (TO) for fluorescence detection. Due to its low cost and favorable safety profile, thiazole orange may see particular benefit in undergraduate teaching labs and research labs performing molecular biology, especially ligations and cloning.
Ethidium bromide remains the most common dye for detection of DNA in agarose gels. This is primarily because it can be obtained very inexpensively and only requires excitation with UV light for detection. Both ethidium bromide and thiazole orange are inexpensive, with low detection limits (1-2 ng/lane)1. There are two main drawbacks to ethidium bromide, however, that thiazole orange improves upon.&#160;
First, ethidium bromide is a mutagen2 with special handling, shipping, and disposal requirements, whereas thiazole orange is less mutagenic (3&#8211;4x less mutagenic in Ames test)3,4 and can be generally disposed of with common chemical waste.
Second, ethidium bromide requires UV light for detection. Thiazole orange can similarly use UV light if desired, but can also be detected with blue light. UV light, while commonly used, has a few salient disadvantages. First, it is damaging to human skin and eyes. While UV light can be used safely by trained professionals, accidental skin or eye damage (functionally similar to sunburns) from laboratory UV light are not uncommon particularly with inexperienced scientists. Second, UV light is extremely damaging to DNA samples5, which reduces the success of downstream experiments (such as ligation and transformation)1,6,7. TO allows detection with blue light (&#955;ex,max = 510 nm (488 nm and 470 nm also show strong excitation)), which does not cause skin damage or DNA damage (although any intense light may still be harmful to eyes), greatly decreasing the risks to both the scientist and the sample.&#160;
TO is not the only fluorescent dye alternative to ethidium bromide; its advantage is cost. TO was discovered in the 1980s as a reticulocyte stain8, and has found utility in a number of DNA-based fluorescence experiments9,10,11,12,13. It is currently sold by multiple suppliers. TO is the parent compound of additional, more expensive, blue-light&#8211;detectable commercial dyes, and behaves similarly during electrophoresis, using UV or blue light for detection1. Furthermore, while other dyes are more sensitive to very low DNA concentrations than either EtBr or TO, for generic electrophoresis experiments, such dyes are prohibitively expensive in many contexts.&#160;

<table><tbody><tr><td>2-log DNA ladder</td><td>New England Biolabs</td><td>N0469S</td><td></td></tr><tr><td>Agarose (Genetic Analysis Grade)</td><td>Fisher</td><td>BP1356-100</td><td></td></tr><tr><td>Blue-light flashlight</td><td>WAYLLSHINE (Amazon)</td><td>WAYLLSHINE Scalable Blue LED</td><td></td></tr><tr><td>ChemiDoc MP</td><td>Biorad</td><td>1708280</td><td></td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>D8418</td><td></td></tr><tr><td>ethidium bromide</td><td>Fisher</td><td>BP1302-10</td><td>For comparison, not necessary for protocol</td></tr><tr><td>Gel apparatus (Owl Easy Cast)</td><td>Thermo Scientific</td><td>B1A</td><td></td></tr><tr><td>Qiagen Qiaquick Gel extraction kit</td><td>Qiagen</td><td>28704</td><td></td></tr><tr><td>Safe Imager Viewing Glasses</td><td>Invitrogen</td><td>S37103</td><td>Necessary for using blue light flashlight.*</td></tr><tr><td>SafeImager 2.0 (Blue light transilluminator)</td><td>Invitrogen</td><td>G6600</td><td>Blue light flashlight may be used as alternative</td></tr><tr><td>SYBR Safe</td><td>Invitrogen</td><td>S33102</td><td>For comparison, not necessary for protocol</td></tr><tr><td>TAE (Tris-Acetate-EDTA)</td><td>Corning</td><td>46-010-CM</td><td></td></tr><tr><td>Thiazole orange</td><td>Sigma-Aldrich</td><td>390062</td><td></td></tr><tr><td colspan="4">*Glasses are also included with Invitrogen G6600</td></tr></tbody></table>

dna electrophoresis using thiazole orange instead of ethidium bromide or alternative dyes

DNA gel electrophoresis using agarose is a common tool in molecular biology laboratories, allowing separation of DNA fragments by size. After separation, DNA is visualized by staining. This article demonstrates how to use thiazole orange to stain DNA. Thiazole orange compares favorably to common staining methods, in that it is sensitive, inexpensive, excitable with UV or blue light (to prevent sample damage), and safer than ethidium bromide. Labs already equipped to run DNA electrophoresis experiments using ethidium bromide can generally switch dyes with no additional changes to existing protocols, using UV light for detection. Blue-light detection to avoid sample damage can additionally be achieved with a blue-light source and emission filter. Labs already equipped for blue-light detection can simply switch dyes with no additional changes to existing protocols.

Thiazole orange enables detection of DNA, without using ethidium bromide and without using DNA-damaging UV light. Ethidium bromide is well-known to be mutagenic, so eliminating it from the lab may be advantageous. UV light damages DNA and lowers transformation efficiency significantly, whereas blue light does not damage DNA. Detection limits are similar between ethidium bromide, thiazole orange, and a common, blue-light&#8211;detectable commercial DNA dye (Figure 1, see Table of Mate...

Watch this Scientific Journal Video about DNA Electrophoresis Using Thiazole Orange Instead of Ethidium Bromide or Alternative Dyes at JoVE.com

DNA Electrophoresis Using Thiazole Orange Instead of Ethidium Bromide or Alternative Dyes

Here, we present a protocol to use thiazole orange for the detection of DNA in gel electrophoresis experiments. The use of thiazole orange allows elimination of ethidium bromide, and fluorescence detection can be achieved with either UV or blue light.

DNA gel electrophoresis using agarose is a common tool in molecular biology laboratories, allowing separation of DNA fragments by size. After separation, ...

dna-electrophoresis-using-thiazole-orange-instead-ethidium-bromide-or

Research

JoVE Journal

Biochemistry

13.8K Views.  Christopher Newport University. Here, we present a protocol to use thiazole orange for the detection of DNA in gel electrophoresis experiments. The use of thiazole orange allows elimination of ethidium bromide, and fluorescence detection can be achieved with either UV or blue light.

Video: DNA Electrophoresis Using Thiazole Orange Instead of Ethidium Bromide or Alternative Dyes

An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. Radioactivity has been the predominant method of DNA labeling in EMSAs. However, recent advances in fluorescent dyes and scanning methods have prompted the use of fluorescent tagging of DNA as an alternative to radioactivity for the advantages of easy handling, saving time, reducing cost, and improving safety. We have recently used fluorescent EMSA (fEMSA) to successfully address an important biological question. Our fEMSA analysis provides mechanistic insight into the effect of a missense mutation, G73E, in the highly conserved HMG transcription factor SOX-2 on olfactory neuron type diversification. We found that mutant SOX-2G73E protein alters specific DNA binding activity, thereby causing olfactory neuron identity transformation. Here, we present an optimized and cost-effective step-by-step protocol for fEMSA using infrared fluorescent dye-labeled oligonucleotides containing the LIM-4/SOX-2 adjacent target sites and purified SOX-2 proteins (WT and mutant SOX-2G73E proteins) as a biological example.

We describe here an optimized protocol of fluorescent Electrophoretic Mobility Shift Assays (fEMSA) using purified SOX-2 proteins together with infrared fluorescent dye-labeled DNA probes as a case study to tackle an important biological question.

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. ...

DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis can be characterized using an in vitro DNA synthesis assay followed by polyacrylamide gel electrophoresis. The goal of this assay is to quantify synthesis of both natural DNA and modified DNA (M-DNA). These approaches are particularly useful for resolving oligonucleotides with single nucleotide resolution, enabling observation of individual steps during enzymatic oligonucleotide synthesis. These methods have been applied to the evaluation of an array of biochemical and biophysical properties such as the measurement of steady-state rate constants of individual steps of DNA synthesis, the error rate of DNA synthesis, and DNA binding affinity. By using modified components including, but not limited to, modified nucleoside triphosphates (NTP), M-DNA, and/or mutant DNA polymerases, the relative utility of substrate-DNA polymerase pairs can be effectively evaluated. Here, we detail the assay itself, including the changes that must be made to accommodate nontraditional primer DNA labeling strategies such as near-infrared fluorescently labeled DNA. Additionally, we have detailed crucial technical steps for acrylamide gel pouring and running, which can often be technically challenging.

This protocol describes the characterization of DNA polymerase synthesis of modified DNA through observation of changes to near-infrared fluorescently labeled DNA using gel electrophoresis and gel imaging. Acrylamide gels are used for high resolution imaging of the separation of short nucleic acids, which migrate at different rates depending on size.

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis ...

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are expressed in spatial relation to each other in situ. Multi-target chromogenic whole-mount in situ hybridization (MC-WISH) greatly facilitates the instant comparison of gene expression patterns, as it allows distinctive visualization of different mRNA species in contrasting colors in the same sample specimen. This provides the possibility to relate gene expression domains topographically to each other with high accuracy and to define unique and overlapping expression sites. In the presented protocol, we describe a MC-WISH procedure for comparing mRNA expression patterns of different genes in Drosophila embryos. Up to three RNA probes, each specific for another gene and labeled by a different hapten, are simultaneously hybridized to the embryo samples and subsequently detected by alkaline phosphatase-based colorimetric immunohistochemistry. The described procedure is detailed here for Drosophila, but works equally well with zebrafish embryos.

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

We describe a multi-target chromogenic whole-mount in situ hybridization (MC-WISH) procedure in intact Drosophila embryos allowing simultaneous and specific detection of three different mRNA distribution patterns by contrasting color precipitates.

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are ...

Developmental Biology

DNA Staining Method Based on Formazan Precipitation Induced by Blue Light Exposure

DNA staining methods are very important for biomedical research. We designed a simple method that allows DNA visualization to the naked eye by the formation of a colored precipitate. It works by soaking the acrylamide or agarose DNA gel in a solution of 1x (equivalent to 2.0 &#181;M) SYBR Green I (SG I) and 0.20 mM nitro blue tetrazolium that produces a purple precipitate of formazan when exposed to sunlight or specifically blue light. Also, DNA recovery tests were performed using an ampicillin resistant plasmid in an agarose gel stained with our method. A larger number of colonies was obtained with our method than with traditional staining using SG I with ultraviolet illumination. The described method is fast, specific, and non-toxic for DNA detection, allowing visualization of biomolecules to the "naked eye" without a transilluminator, and is inexpensive and appropriate for field use. For these reasons, our new DNA staining method has potential benefits to both research and industry.

This method allows selective staining and quantification of DNA in gels by soaking the gel in a SYBR Green I/Nitro Blue Tetrazolium solution and then exposing it to sunlight or a blue light source. This produces a visible precipitate and requires almost no equipment, making it ideal for field use.

DNA staining methods are very important for biomedical research. We designed a simple method that allows DNA visualization to the naked eye by the ...

Determining if DNA Stained with a Cyanine Dye Can Be Digested with Restriction Enzymes

Visualization of DNA for fluorescence microscopy utilizes a variety of dyes such as cyanine dyes. These dyes are utilized due to their high affinity and sensitivity for DNA. In order to determine if the DNA molecules are full length after the completion of the experiment, a method is required to determine if the stained molecules are full length by digesting DNA with restriction enzymes. However, stained DNA may inhibit the enzymes, so a method is needed to determine what enzymes one could use for fluorochrome stained DNA. In this method, DNA is stained with a cyanine dye overnight to allow the dye and DNA to equilibrate. Next, stained DNA is digested with a restriction enzyme, loaded into a gel and electrophoresed. The experimental DNA digest bands are compared to an in silico digest to determine the restriction enzyme activity. If there is the same number of bands as expected, then the reaction is complete. More bands than expected indicate partial digestion and less bands indicate incomplete digestion. The advantage of this method is its simplicity and it uses equipment that a scientist would need for a restriction enzyme assay and gel electrophoresis. A limitation of this method is that the enzymes available to most scientists are commercially available enzymes; however, any restriction enzymes could be used.

Staining DNA molecules for fluorescence microscopy allows a scientist to view them during an experiment. In the method presented here, DNA molecules are pre-stained with fluorescent dyes and digested with methylation and non-methylation sensitive restriction enzymes.

Visualization of DNA for fluorescence microscopy utilizes a variety of dyes such as cyanine dyes. These dyes are utilized due to their high affinity and ...

Detection of Bacteria Using Fluorogenic DNAzymes

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses each and every year. Prevention of such outbreaks and minimization of the impact of an ongoing epidemic place an ever-increasing demand for analytical methods that can accurately identify culprit pathogens at the earliest stage. Although there is a large array of effective methods for pathogen detection, none of them can satisfy all the following five premier requirements embodied for an ideal detection method: high specificity (detecting only the bacterium of interest), high sensitivity (capable of detecting as low as a single live bacterial cell), short time-to-results (minutes to hours), great operational simplicity (no need for lengthy sampling procedures and the use of specialized equipment), and cost effectiveness. For example, classical microbiological methods are highly specific but require a long time (days to weeks) to acquire a definitive result.1 PCR- and antibody-based techniques offer shorter waiting times (hours to days), but they require the use of expensive reagents and/or sophisticated equipment.2-4 Consequently, there is still a great demand for scientific research towards developing innovative bacterial detection methods that offer improved characteristics in one or more of the aforementioned requirements. Our laboratory is interested in examining the potential of DNAzymes as a novel class of molecular probes for biosensing applications including bacterial detection.5
DNAzymes (also known as deoxyribozymes or DNA enzymes) are man-made single-stranded DNA molecules with the capability of catalyzing chemical reactions.6-8 These molecules can be isolated from a vast random-sequence DNA pool (which contains as many as 1016 individual sequences) by a process known as "in vitro selection" or "SELEX" (systematic evolution of ligands by exponential enrichment).9-16 These special DNA molecules have been widely examined in recent years as molecular tools for biosensing applications.6-8
Our laboratory has established in vitro selection procedures for isolating RNA-cleaving fluorescent DNAzymes (RFDs; Fig. 1) and investigated the use of RFDs as analytical tools.17-29 RFDs catalyze the cleavage of a DNA-RNA chimeric substrate at a single ribonucleotide junction (R) that is flanked by a fluorophore (F) and a quencher (Q). The close proximity of F and Q renders the uncleaved substrate minimal fluorescence. However, the cleavage event leads to the separation of F and Q, which is accompanied by significant increase of fluorescence intensity.
More recently, we developed a method of isolating RFDs for bacterial detection.5 These special RFDs were isolated to "light up" in the presence of the crude extracellular mixture (CEM) left behind by a specific type of bacteria in their environment or in the media they are cultured (Fig. 1). The use of crude mixture circumvents the tedious process of purifying and identifying a suitable target from the microbe of interest for biosensor development (which could take months or years to complete). The use of extracellular targets means the assaying procedure is simple because there is no need for steps to obtain intracellular targets.
Using the above approach, we derived an RFD that cleaves its substrate (FS1; Fig. 2A) only in the presence of the CEM produced by E. coli (CEM-EC).5 This E. coli-sensing RFD, named RFD-EC1 (Fig. 2A), was found to be strictly responsive to CEM-EC but nonresponsive to CEMs from a host of other bacteria (Fig. 3).
Here we present the key experimental procedures for setting up E. coli detection assays using RFD-EC1 and representative results.

We have recently reported a novel approach for generating fluorogenic DNAzyme probes that can be applied to set up a simple, "mix-and-read" fluorescent assay for bacterial detection. These special DNA probes catalyze the cleavage of a chromophore-modified DNA-RNA chimeric substrate in the presence of crude extracellular mixture (CEM) produced by a specific bacterium, thereby translating bacterial detection into fluorescence signal generation. In this report we will describe key experimental procedures where a specific DNAzyme probe denoted "RFD-EC1" is employed for the detection of the model bacterium, Escherichia coli (E. coli).

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses ...

Immunology and Infection

Fluorescent Visualization of Mango-tagged RNA in Polyacrylamide Gels via a Poststaining Method

Native and denaturing polyacrylamide gels are routinely used to characterize ribonucleoprotein (RNP) complex mobility and to measure RNA size, respectively. As many gel-imaging techniques use nonspecific stains or expensive fluorophore probes, sensitive, discriminating, and economical gel-imaging methodologies are highly desirable. RNA Mango core sequences are small (19&#8211;22 nt) sequence motifs that, when closed by an arbitrary RNA stem, can be simply and inexpensively appended to an RNA of interest. These Mango tags bind with high affinity and specificity to a thiazole-orange fluorophore ligand called TO1-Biotin, which becomes thousands of times more fluorescent upon binding. Here we show that Mango I, II, III, and IV can be used to specifically image RNA in gels with high sensitivity. As little as 62.5 fmol of RNA in native gels and 125 fmol of RNA in denaturing gels can be detected by soaking gels in an imaging buffer containing potassium and 20 nM TO1-Biotin for 30 min. We demonstrate the specificity of the Mango-tagged system by imaging a Mango-tagged 6S bacterial RNA in the context of a complex mixture of total bacterial RNA.

Here we present a sensitive, rapid, and discriminating post-gel staining method to image RNAs tagged with RNA Mango aptamers I, II, III, or IV, using either native or denaturing polyacrylamide gel electrophoresis (PAGE) gels. After running standard PAGE gels, Mango-tagged RNA can be easily stained with TO1-Biotin and then analyzed using commonly available fluorescence readers.

Native and denaturing polyacrylamide gels are routinely used to characterize ribonucleoprotein (RNP) complex mobility and to measure RNA size, ...

Agarose Gel Electrophoresis for the Separation of DNA Fragments

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits2. During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel&#39;s molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight3. The leading model for DNA movement through an agarose gel is &#34;biased reptation&#34;, whereby the leading edge moves forward and pulls the rest of the molecule along4. The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation5; 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: 
 
1. Understand the mechanism by which DNA fragments are separated within a gel matrix 
2. Understand how conformation of the DNA molecule will determine its mobility through a gel matrix 
3. Identify an agarose solution of appropriate concentration for their needs 
4. Prepare an agarose gel for electrophoresis of DNA samples 
5. Set up the gel electrophoresis apparatus and power supply 
6. Select an appropriate voltage for the separation of DNA fragments 
7. Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands 
8. Determine the sizes of separated DNA fragments 
&#160;

A basic protocol for the separation of DNA fragments using agarose gel electrophoresis is described.

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from ...

Biology

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA)

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical disease or phenotype. For many diseases and traits, most variants are non-coding, and are thus likely to act by impacting subtle, comparatively hard to predict mechanisms controlling gene expression. Here, we describe a general strategic approach to prioritize non-coding variants, and screen them for their function. This approach involves computational prioritization using functional genomic databases followed by experimental analysis of differential binding of transcription factors (TFs) to risk and non-risk alleles. For both electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genetic variants, a synthetic DNA oligonucleotide (oligo) is used to identify factors in the nuclear lysate of disease or phenotype-relevant cells. For EMSA, the oligonucleotides with or without bound nuclear factors (often TFs) are analyzed by non-denaturing electrophoresis on a tris-borate-EDTA (TBE) polyacrylamide gel. For DAPA, the oligonucleotides are bound to a magnetic column and the nuclear factors that specifically bind the DNA sequence are eluted and analyzed through mass spectrometry or with a reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot analysis. This general approach can be widely used to study the function of non-coding genetic variants associated with any disease, trait, or phenotype.

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay EMSA and DNA-affinity Precipitation Assay DAPA

We present a strategic plan and protocol for identifying non-coding genetic variants affecting transcription factor (TF) DNA binding. A detailed experimental protocol is provided for electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genotype-dependent TF DNA binding.

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical ...

Colorimetric Detection of Bacteria Using Litmus Test

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. Such methods need to be user-friendly and produce reliable results that can be easily interpreted by both specialists and non-professionals. The litmus test for pH is simple, quick, and effective as it reports the pH of a test sample via a simple color change. We have developed an approach to take advantage of the litmus test for bacterial detection. The method exploits a bacterium-specific RNA-cleaving DNAzyme to achieve two functions: recognizing a bacterium of interest and providing a mechanism to control the activity of urease. Through the use of magnetic beads immobilized with a DNAzyme-urease conjugate, the presence of bacteria in a test sample is relayed to the release of urease from beads to solution. The released urease is transferred to a test solution to hydrolyze urea into ammonia, resulting in an increase of pH that can be visualized using the classic litmus test.

We describe a protocol for colorimetric detection of E. coli using a modified litmus test that takes advantage of an RNA-cleaving DNAzyme, urease, and magnetic beads.

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. ...

DNA Electrophoresis Using Thiazole Orange Instead of Ethidium Bromide or Alternative Dyes

Summary

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Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

DNA Staining Method Based on Formazan Precipitation Induced by Blue Light Exposure

Determining if DNA Stained with a Cyanine Dye Can Be Digested with Restriction Enzymes

Detection of Bacteria Using Fluorogenic DNAzymes

Fluorescent Visualization of Mango-tagged RNA in Polyacrylamide Gels via a Poststaining Method

Agarose Gel Electrophoresis for the Separation of DNA Fragments

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA)

Colorimetric Detection of Bacteria Using Litmus Test