This research was funded by the National Institute for General Medical Science (NIGMS) (5605100122001), a component of the National Institutes of Health (NIH), as well as University of Nebraska at Kearney (UNK) Summer Student Research Program (SSRP), and UNK Undergraduate Research Fellowship (URF).

1. Preparation of Dyes, Buffers and Agarose Gel
<ol>
	<li>Prepare the following solutions for staining DNA or the digestion of stained DNA.
	<ol>
		<li>Prepare 1x TE (Tris-HCl and ethylenediaminetetraacetic acid; EDTA) buffer using 10 mM Tris-HCl and 1 mM EDTA in a graduated cylinder or volumetric flask. Store at room temperature (18 - 25 &#176;C).</li>
		<li>Make aliquots of each dye: TOTO-1, YOYO-1, POPO-1, BOBO-1, TOTO-3, YOYO-3, POPO-3, BOBO-3 (Table 1). Pipette 4 &#181;L of 1 mM dye into a dark amber or black 1.5 mL tube and add 36 &#181;L of 1x TE, mix using the pipette; this concentration makes a 100 &#181;M dye solution (40 &#181;L total). Complete this step for each dye. Store at 4 &#176;C. 
		NOTE: Avoid exposing dye to light to prevent photobleaching or degradation of the dye.</li>
		<li>Prepare buffer 1 using 10 mM Bis-Tris-Propane-HCl, 10 mM MgCl2, and 100 &#181;g/mL bovine serum albumin (BSA). Prepare buffer 2 using 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, and 100 &#181;g/mL BSA. Prepare buffer 3 using 50 mM Potassium Acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, and 100 &#181;g/mL BSA. Each enzyme will require one of these specific buffers to function.</li>
	</ol>
	</li>
	<li>Prepare the following solutions for gel electrophoresis.
	<ol>
		<li>Make a 6x loading dye by combining 0.25% bromophenol blue, 0.25% xylene cyanol, 15% Ficoll in dH2O. Store at room temperature.</li>
		<li>To make a 0.7% agarose gel, weigh out 0.07 g of high gelling temperature (HGT) agarose in an Erlenmeyer flask, add 100 mL of 1x TAE buffer and place an inverted beaker on top of the flask.
		<ol>
			<li>Heat the solution on a hot plate until bubbles begin to form on the bottom of the flask and float to the top.</li>
			<li>Remove the solution from the hot plate, let the solution cool until the flask can be touched with a bare hand, and then pour the gel into a 24.5 cm x 21.8 cm gel mold with a gel comb to create wells.</li>
			<li>Remove bubbles near the comb or on the surface of the gel by popping them with a pipette tip and allow the gel to solidify for at least 15 minutes. This can be stored in a fridge (4 &#176;C) wrapped in plastic wrap for 1 week to prevent drying of the gel or contamination. For more information on how to run a gel refer to Lee et al.33. 
			NOTE: Use a gel comb with 30 individual wells. Each well should be 4.5 mm wide with a depth of 2 mm. The height of the well will depend on the amount of gel poured into the box.</li>
		</ol>
		</li>
		<li>Prepare 1x TAE (Tris - acetate - EDTA) buffer using 40 mM Tris-HCl, 20 mM acetic acid, and 1 mM EDTA. Store at room temperature (18 - 25 &#176;C).</li>
	</ol>
	</li>
</ol>
2. Preparation of Stained DNA
<ol>
	<li>Stain lambda DNA (300-800 ng) using different aliquots of dyes. Each dye (Table 1) will have six different concentrations tested, for a total of 48 reactions per restriction enzyme. The following process outlines a set up for 1 reaction (Figure 1).
	<ol>
		<li>Dilute lambda DNA using 1x TE to 100 ng/&#181;L. Store in 4 &#176;C. Make a solution with total volume of 300 &#181;L.</li>
		<li>Stain unmethylated lambda DNA. For each band expected from the digest, estimate 75 - 100 ng/band. Add appropriate amounts of dye to produce concentrations of 1.9 &#181;M, 3.8 &#181;M, 19.4 &#181;M, 32.2 &#181;M, 48.3 &#181;M, 63.5 &#181;M / 100 ng of DNA.</li>
		<li>Incubate overnight (15 h - 18 h) at 4 &#176;C.</li>
		<li>Leave one reaction unstained to act as a control25. 
		NOTE: Use unmethylated DNA for the digestion reaction. Methylation sensitive enzymes cannot cut methylated regions in DNA and will give an appearance of an incomplete digestion.</li>
	</ol>
	</li>
</ol>
3. Preparation of the Restriction Enzyme Assay
<ol>
	<li>Digest stained DNA with a restriction enzyme to determine if that enzyme can digest prestained DNA (Figure 1).
	<ol>
		<li>The following day, add 20 U of restriction enzyme, 3 &#181;L of the appropriate buffer ( Table 2), and distilled, autoclaved and filtered water for a total of 30 &#181;L. For each enzyme reaction, choose the buffer with the highest cutting efficiency, which can be found on the manufacturer&#39;s website.</li>
		<li>Place the reactions in a water bath at the temperature of optimal enzymatic activity, listed in Table 2 for the 6 enzymes used in Maschmann et al., for 2 - 4 h (Table 2)25.</li>
		<li>Stop the reaction by adding 2 &#181;L of 0.5 M EDTA pH 8.0.</li>
	</ol>
	</li>
</ol>
4. Electroeluting Stained DNA in an Agarose Gel
<ol>
	<li>Remove the sides from the 24.5 cm x 21.8 cm gel mold (Step 1.2.2) and place the gel into the gel electrophoresis box. Pour 2.5 L of 1x TAE buffer into the box. Carefully remove the gel comb from the gel, so the wells are accessible33.</li>
	<li>Pipette the restriction enzyme reactions onto a plastic film and combine each reaction with 6x loading dye. For each reaction solution, add 1 &#181;L of 6x loading dye per 5 - 6 &#181;L of DNA solution (1x). Then pipette the reactions (containing the loading dye, &#60;18 &#181;L) into the wells.</li>
	<li>Mix 1 &#181;L of 1 kb ladder, 9 &#181;L of 1x TE, and 2 &#181;L of 6x loading dye. Load solution into the wells that flank the other solutions.</li>
	<li>Cover the gel box with dark blue or black paper or fabric. Connect the gel box to a power supply, set the power supply to 30 V, and run it overnight (15 - 20 h). Turn off the lights while the gel is running, to prevent photocleavage of the labeled DNA.</li>
	<li>The next morning turn off the power supply. Take the gel out using the tray and transfer it to a container with 45 &#181;L ethidium bromide and 1 L of 1x TAE buffer. Store the container at room temperature in the dark or cover in foil to prevent exposure to light. Let gel stain for at least 45 min at room temperature. 
	CAUTION: Use ethidium bromide with gloves and wear safety glasses. When cleaning out the container containing ethidium bromide solution and throwing away the gel, consult your states disposal guidelines to determine how to deal with used ethidium bromide; additionally, other stains may be utilized instead of ethidium bromide.</li>
</ol>
5. Imaging the Agarose Gel
<ol>
	<li>Place the gel on top of a blue light transilluminator, which emits maximum light output between 400-500 nm. Take pictures with the camera attached to the station. 
	CAUTION: Make sure to use the cover for the illuminator and put on protective glasses prior to turning on the light source. 
	NOTE: This step may also be completed using any UV light source or standard gel scanner.</li>
	<li>View the pictures in ImageJ by dragging the file to the ImageJ status bar and the image will pop up.</li>
	<li>Determine the digestion rate for the experiment by comparing the bands on the gel to the expected in silico gel pattern. To determine the digestion rate, the number of experimental bands is divided by the expected number of DNA bands24,25. If the experimental digested DNA band pattern has the expected pattern, the digestion rate is 1. If the pattern has more fragments than expected, the digestion rate is greater than 1. If the pattern has less fragments than expected, the digestion rate is less than one.</li>
</ol>

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The authors have nothing to disclose.

In order to digest fluorescently labeled DNA (Figure 1), a series of steps are required. First, DNA is stained with a fluorochrome overnight. DNA can be incubated with cyanine dimers for a shorter period of time; however, Carlsson et al. found that DNA created double bands for each DNA size due to incomplete staining20. To remedy this, the DNA can be stained overnight to prevent double bands from occurring. This is a critical step in the protocol. If the dye is not incubated for a long enough time, the dye-DNA may not be at equilibrium and will give a double-banding pattern. If this does occur, allow the DNA-dye mixture to equilibrate for a longer period of time.
Next, a restriction enzyme is added to the DNA-dye mixture and digested for 2 - 4 h at a specific temperature for each enzyme. The DNA must be unmethylated for methylation sensitive enzymes. If it is not, methylated DNA will inhibit the restriction enzyme from cutting DNA in methylated regions. Additionally, the reaction must be incubated in the correct buffer and temperature for the enzyme. Each enzyme has a list of buffers and the enzyme activity in each buffer. Choose the buffer with enzyme activity at 100%, if possible. As for temperature, make sure it is incubated in the correct temperature. If it is not, it may cause the enzyme to lose activity or denature if the temperature is too high. EDTA is utilized to stop the reaction. The company where the enzymes were purchased will have a chart with the optimal temperatures and buffer conditions for that enzyme. If the control does not give the correct number of bands at the right molecular weights, check to see if the enzyme is expired, increase the incubation time, or check to make sure the temperature is correct for that enzyme.
Once the reaction is completed, the samples can be loaded into a gel. First, a gel is loaded into the gel container and immersed in 1x TAE buffer. Next, the digested DNA is mixed with loading dye and the solution is loaded into the wells of the gel. The lid is slid over and the wires are attached to the gel box. A dark paper, that is taped together, is placed on top of the lid to cover the lid and drape over the sides. Another critical step is that the samples are protected from the light at all times, especially while the gel is running. The power supply should be turned on and the lights to the room turned off. In order to prevent photocleavage of DNA, samples are protected from light the whole time. If there is no dark paper, a dark fabric can be used as well. If the DNA does not move, make sure the wires are connected to the box and the run button has been pressed.
In the morning, take the gel out of the gel box and load the gel into a container that contains ethidium bromide. Ethidium bromide allows us to see the DNA bands under the blue light transilluminator (Figure 2). Other dyes can be used in place of ethidium bromide. A camera can be mounted above the blue light transilluminator to capture pictures of the gel. Depending on the setup, an imaging station can also be utilized.
This method can be utilized for any dye that intercalates or stains DNA, not just the TOTO series. Maschmann et al. modified a technique developed by Meng et al. to determine if fluorescently stained DNA could be digested with restriction enzymes24,25. This is the only technique utilized to determine if pre-stained DNA affects restriction enzymes. This method is limited to the restriction enzymes commercially available, unless a scientist has access to other restriction enzymes that are not listed commercially. Depending on DNA, the enzyme chosen should have a discernable DNA band pattern and enough bands (&#62;3) to determine if the DNA molecule is full length. Also, the DNA should be unmethylated if methylation sensitive enzymes are used. If DNA is methylated, then do not use methylation sensitive enzymes.
This method is an easy to use way to determine if restriction enzymes can cut DNA molecules at the recognition site stained with an intercalating dye without needing to design primers for a given cut site34. The future applications of this method would be to determine if fluorescently labeled molecules from an experiment were full length by using a restriction enzyme and determining the size of the DNA bands from the digest or to determine the methylation profiles of DNA molecules. Future experiments could also determine if nicking enzymes are affected by the cyanine dyes or other fluorescent dyes.

The TOTO series (TOTO-1, YOYO-1, POPO-1, BOBO-1, TOTO-3, YOYO-3, POPO-3, and BOBO-3; Table 1) is utilized in a wide variety of experiments where the visualization of DNA is required1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17. The cyanine dimer family is widely used due to their quantum yield, sensitivity, and high affinity for DNA molecules18,19,20. Cyanine dimer dyes have great selectivity for double stranded DNA and when intercalated have a 100 to 1000 fold increase of fluorescence21. Pyridinium dyes (YOYO-1, TOTO-1, YOYO-3, and TOTO-3) have a shorter emission wavelength than their quinolium dye (BOBO-1, POPO-1, BOBO-3, and POPO-3) counterparts (Table 1)22. Also, the quantum yield for cyanine dimers intercalated into DNA is high (0.2 - 0.6)22. However, using an enzyme to determine the methylation profile of a DNA molecule2 or stretch23 of a DNA molecule already stained with fluorescent dye requires a method to determine what enzymes will digest stained DNA. Any type of dye that intercalates into DNA or any enzyme that gives a discernable pattern of the DNA substrate can be used for this method.
Meng et al. first determined the digestion rate of prestained DNA through gel electrophoresis using a variety of different dyes24. Maschmann et al. delved in deeper to look at the TOTO family of dyes. Both determined the digestion rate of stained DNA to see if DNA stained with a given dye could be digested with a restriction enzyme25. Other methods study binding effects of dyes intercalated with DNA using optical tweezers26 or NMR27. Either method requires specialized equipment; whereas, this method allows equipment that most molecular biology labs have to determine if a dye interferes with a restriction enzyme digestion.
Additionally, in other methods to measure the length of a given molecule, optical mapping has elongated unstained DNA molecules on a surface and digested DNA to determine the stretch and size of fragments. Intercalation of dye has been shown to increase the contour length of fluorescently stained DNA molecules and depending on the dye used, the contour lengths are different21. This method has been utilized in a variety of genomes1,3,4,6,13,28,29,30,31. However, if molecules were pre-stained, depending on the dye and enzyme, the enzyme may not be able to cut DNA stained with a given dye. Therefore, this method determines if DNA stained with a given dye can be digested with an enzyme. Additionally, depending on the concentration of the dye and the dye utilized, the mobility of the DNA bands in a gel will migrate more slowly than native DNA due to the partial unwinding of the DNA backbone to make room for the dye to insert between base pairs32.
However, sometimes these dyes can partially or completely inhibit the action of certain restriction enzymes7,24. This is thought to be due to a structural change in DNA caused by the attachment of the fluorescent dye, which may prevent the enzyme from recognizing its specific sequence. Understanding how these dyes affect restriction enzymes can help in experiments where the methylation profile or the stretch of stained DNA is required.
In our method, DNA was stained with a fluorochrome of interest and digested with a restriction enzyme. Then DNA was electrophoresed on a gel, imaged, and the restriction enzyme digestion rate was measured. The restriction enzymes were chosen based on the cut pattern on a gel. Too many bands caused overlap of DNA bands and too few bands did not give a complete picture of the DNA molecule. There is a sweet spot to be able to determine the profile of the digested DNA molecule; therefore, it will depend on the DNA used and the enzyme. An advantage of this method is its simplicity; it only requires equipment used in a restriction digestion and gel electrophoresis.

<table border="0" cellpadding="0" cellspacing="0" width="605">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">Unmethylated lambda DNA</td>
			<td style="width:111px;">NEB</td>
			<td style="width:155px;">N3013S</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">YOYO-1</td>
			<td style="width:111px;">ThermoFisher Scientific</td>
			<td>Y3601</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">TOTO-1</td>
			<td style="width:111px;">ThermoFisher Scientific</td>
			<td>T3600</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">POPO-1</td>
			<td style="width:111px;">ThermoFisher Scientific</td>
			<td>P3580</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">BOBO-1</td>
			<td style="width:111px;">ThermoFisher Scientific</td>
			<td>B3582</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">YOYO-3</td>
			<td style="width:111px;">ThermoFisher Scientific</td>
			<td>Y3606</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">TOTO-3</td>
			<td style="width:111px;">ThermoFisher Scientific</td>
			<td>T3604</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">POPO-3</td>
			<td style="width:111px;">ThermoFisher Scientific</td>
			<td>P3584</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">BamHI</td>
			<td style="width:111px;">NEB</td>
			<td>R0136S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">PmlI</td>
			<td style="width:111px;">NEB</td>
			<td>R0532S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">ScaI</td>
			<td style="width:111px;">NEB</td>
			<td>R3122S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">EcoRI&#160;</td>
			<td style="width:111px;">NEB</td>
			<td>R0101S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">HindIII</td>
			<td style="width:111px;">NEB</td>
			<td>R0104S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">SmaI</td>
			<td style="width:111px;">NEB</td>
			<td>R0141S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">Tris</td>
			<td style="width:111px;">Fisher</td>
			<td>BP152-1</td>
			<td>Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">EDTA</td>
			<td style="width:111px;">Fisher</td>
			<td style="width:155px;">S316-212</td>
			<td>Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">HCl</td>
			<td style="width:111px;">Fisher</td>
			<td>S25358</td>
			<td>Corrosive and irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">Buffer 1</td>
			<td style="width:111px;">NEB</td>
			<td>B7201S</td>
			<td>NEB 1.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">Buffer 2</td>
			<td style="width:111px;">NEB</td>
			<td>B7202S</td>
			<td>NEB 2.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">Buffer 3</td>
			<td style="width:111px;">NEB</td>
			<td>B7204S</td>
			<td>NEB Cutsmart</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">High gelling temperature agarose gel</td>
			<td style="width:111px;">Lonza</td>
			<td>50041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">Acetic Acid</td>
			<td style="width:111px;">Fisher</td>
			<td>S25118</td>
			<td>Hazardous</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">Blue light transilluminator</td>
			<td style="width:111px;">Dark Reader</td>
			<td>DR196</td>
			<td>Wear correct eyewear&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:173px;">Ethidium bromide</td>
			<td style="width:111px;">Fisher</td>
			<td>15585011</td>
			<td style="width:167px;">Carcinogen; other alternatives are Nancy 520, Gel red, etc.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">Cannon EOS Rebel T3I camera</td>
			<td style="width:111px;">Cannon</td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Apogee Model H4</td>
			<td style="width:111px;">Biorad</td>
			<td>1704510</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">Power Pac Basic Power Supply</td>
			<td style="width:111px;">Biorad</td>
			<td>1645050&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">Ficoll</td>
			<td style="width:111px;">Fisher Scientific</td>
			<td style="width:155px;">BP525-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">Bromophenol blue</td>
			<td style="width:111px;">Fisher Scientific</td>
			<td style="width:155px;">B-392</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">Xylene Cyanol</td>
			<td style="width:111px;">Eastmen</td>
			<td style="width:155px;">T1579</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

In order to determine if an intercalating dye will affect a restriction enzyme digesting DNA, the correct order of steps must be followed (Figure 1). Once DNA is stained and digested with a given enzyme, a picture of the gel can be taken to determine the number of fragments and their size (Figure 2). In order to determine the enzyme efficiency, the total number of expected visible bands divided by the number of visible bands. Enzyme efficiency equaled unity, if the number of bands expected and seen match. If there are more bands on the gel then expected, the value is greater than one and indicates partially digested reactions. If there are less bands then expected, then the value is less than one, which indicates incomplete digestion. In Figure 2a, lanes 3 and 4 show the mobility of the bands has decreased, causing the bands to shift towards the wells. In lanes 1 and 2, the amount of dye does not affect the mobility noticeably, so the bands match the control. In Figure 2a, lanes 5 and 6 show more bands than the control, so it indicates partial digestion. Which means, the dyes affected the cleavage of the DNA at the recognition site for that enzyme. In Figure 2b, all expected bands are seen for all dye concentrations, so the dye did not interfere with the digestion of DNA. Figure 2c tracks the mobility shifts with colored asterisks.
As dye concentration increases for dyes with -1, the mobility decreases. In order to determine the approximate size of the bands, a control is required to know where the native DNA molecules are expected compared to the stained DNA (Figure 2a). For dyes with -3, the mobility did not decrease to the extent that -1 dyes did, so it was easier to determine the fragment size (Figure 2b). The mobility difference is due to the type of dye utilized, as seen in Maschmann et al.25.The variance of these dyes is seen due to the structures of the dyes. The linker between the aromatic moiety for dyes -3 was longer than -1. The dyes may intercalate slightly differently due to the difference in the linker size, which may explain why the mobility shift is less for -3 dyes.
If there is no DNA, or the DNA bands are very faint in the gel, increasing the concentration of DNA is required. However, if the DNA concentration increases, then the amount of dye required will also increase to keep the correct concentration of dye to DNA ratio.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57141/57141fig1.jpg" /> 
Figure 1: A schematic of the steps required when digesting fluorochrome labeled DNA molecules. DNA and YOYO-1, or another dye from the TOTO series, is added to a black tube and incubated overnight (O/N; 15 - 20 h). A restriction enzyme is added to a tube; it is placed in an incubator, at the preferred temperature, for 2 - 4 h (Table 2). Load samples into an immersed 0.7% agarose gel made with 1x TAE buffer. Dark colored paper covers the gel box and the lights are turned off, while the gel is ran overnight to prevent photocleavage of the dye. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57141/57141fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57141/57141fig2.jpg" /> 
Figure 2: Restriction digestion of DNA intercalated with a cyanine dimer dye. (a) Lambda DNA is stained with different concentrations of YOYO-1 overnight (lane C: digested lambda DNA with no dye (control), lane 1: 1.9 ng, lane 2: 3.8 ng, lane 3: 19.4 ng, lane 4: 32.2 ng, lane 5: 48.3 ng, lane 6: 63.5 ng of dye per 100 ng of DNA) and then digested with EcoRI at 37 &#176;C for 2 - 4 h. All reactions are electrophoresed (E = 0.85 V/cm) on a 0.7% high gelling temperature (HGT) agarose gel made with 1x TAE buffer (1x TAE; 40 mM Tris-HCl, 20 mM Acetic acid, 1 mM EDTA). Gels are stained with ethidium bromide and imaged with a blue light transilluminator coupled to a camera. Lane L is a 1 kb ladder (sizes, kb); lane &#955; is full length lambda DNA; lane E is the expected band pattern (sizes, kb). (b) Lambda DNA is stained with YOYO-3 (lane 1: 1.9 ng, lane 2: 3.8 ng, lane 3: 19.4 ng, lane 4: 32.2 ng, lane 5: 48.3 ng, lane 6: 63.5 ng of dye per 100 ng of DNA) and digested with HindIII at 37 &#176;C. (c) To see how the mobility shifted as the YOYO-1 concentration is increased, colored asterisks are located next to each band for each YOYO-1 concentration. For example, the band at 3.5 kb has a red asterisk next to each band in each YOYO-1 concentration. The unexpected bands in lane 5 and 6 do not have an asterisk next to the bands. The figure is modified from Maschmann et al.25. Copyright (2017) Nucleosides, Nucleotides, and Nucleic Acids. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57141/57141fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Chemical Name</td>
			<td>Abbreviated Name</td>
			<td>Ex/Em (nm)</td>
		</tr>
		<tr>
			<td>[(1&#8211;1&#8242;-[1,3-propanediylbis[(dimethyliminio)-3,1-propanediyl] bis[4-[(3-methyl-2(3H)-benzothiazolylidene)methyl]]-, tetraiodide</td>
			<td>TOTO-1</td>
			<td>514/533</td>
		</tr>
		<tr>
			<td>1,1&#8242; - (4,4,8,8-tetramethyl-4,8-diazaundecamethylene)bis[4-[(3-methylbenzo-1,3-oxazol -2-yl)methylidene]-l,4-dihydroquinolinium] tetraiodide}</td>
			<td>YOYO-1</td>
			<td>491/509</td>
		</tr>
		<tr>
			<td>2,2&#8242;-[1,3-prop-anediylbis[(dimethyliminio)-3,1-propanediyl-1(4H)-pyridinyl-4-ylidenemethylid- yne]]bis[3-methyl]-, tetraiodide</td>
			<td>POPO-1</td>
			<td>434/456</td>
		</tr>
		<tr>
			<td>2,2&#8242;-[1,3-propanediylbis[(dimethylimi- nio)-3,1-propanediyl-1(4H)-pyridinyl-4&#8211;1,1&#8242; -[4,8-bis(dimethyliminio)undecane-1,11-diyl]bis{4-[3-(3-methyl-1,3-benzothiazol-2(3H)-ylidene)prop-1-en-1-yl]qui- nolinium}</td>
			<td>BOBO-1</td>
			<td>462/481</td>
		</tr>
		<tr>
			<td>2,2&#8242;-{[4,8-bis(dimethyliminio)undecane-1,11-diyl]bis[quinolin-1-yl-4- ylideneprop-1-en-1-yl-3-ylidene]}bis(3-methyl-1,3-benzothiazol-3-ium) tetraio- dide</td>
			<td>TOTO-3</td>
			<td>642/660</td>
		</tr>
		<tr>
			<td>1,1&#8242;-[1,3-propanediylbis[(dimethyliminio)-3,1-propanediyl]]bis[4- [3-(3-methyl-2(3H)-benzoxazolylidene)-1-propenyl]]-, tetraiodide</td>
			<td>YOYO-3</td>
			<td>612/631</td>
		</tr>
		<tr>
			<td>2,2&#8242; -[1,3-propanediylbis[(dimethyliminio)-3,1-propanediyl-1(4H)-pyridinyl-4- ylidene-1-pr-open-1-yl-3-ylidene]]bis[3-methyl]-, tetraiodide</td>
			<td>POPO-3</td>
			<td>534/570</td>
		</tr>
		<tr>
			<td>2,2&#39;-{propane-1,3-diylbis[(dimethylazaniumdiyl)propane-3,1-diylpyridin-1-yl-4-ylideneprop-1-en-1-yl-3-ylidene]}bis(3-methyl-1,3-benzothiazol-3-ium) tetraiodide</td>
			<td>BOBO-3</td>
			<td>570/602</td>
		</tr>
	</tbody>
</table>
Table 1: Abbreviated names of TOTO family of dyes with IUPAC names with emission (Em) and excitation (Ex) maximima22.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Enzyme</td>
			<td>Methylation Sensitive</td>
			<td>Buffer</td>
			<td>Water Bath Temperature</td>
		</tr>
		<tr>
			<td>BamHI</td>
			<td>No</td>
			<td>2</td>
			<td>37 &#176;C</td>
		</tr>
		<tr>
			<td>EcoRI</td>
			<td>Yes</td>
			<td>2</td>
			<td>37 &#176;C</td>
		</tr>
		<tr>
			<td>HindIII</td>
			<td>No</td>
			<td>2</td>
			<td>37 &#176;C</td>
		</tr>
		<tr>
			<td>PmlI</td>
			<td>Yes</td>
			<td>1</td>
			<td>37 &#176;C</td>
		</tr>
		<tr>
			<td>ScaI</td>
			<td>No</td>
			<td>2</td>
			<td>37 &#176;C</td>
		</tr>
		<tr>
			<td>SmaI</td>
			<td>Yes</td>
			<td>3</td>
			<td>25 &#176;C</td>
		</tr>
	</tbody>
</table>
Table 2: Listed in the table is a subset of possible enzymes that could be used in these experiments. Three enzymes are methylation sensitive and three are not methylation sensitive. The buffers used for the enzymes and incubation temperatures are listed for each enzyme.

Watch this Scientific Journal Video about Determining if DNA Stained with a Cyanine Dye Can Be Digested with Restriction Enzymes at JoVE.com

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

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 ...

determining-if-dna-stained-with-cyanine-dye-can-be-digested-with

Nanyang Technological University

Research

JoVE Journal

Biochemistry

8.7K Views.  University of Nebraska - Kearney. 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.

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

Highly Sensitive and Rapid Fluorescence Detection with a Portable FRET Analyzer

Recent improvements in F&#246;rster resonance energy transfer (FRET) sensors have enabled their use to detect various small molecules including ions and amino acids. However, the innate weak signal intensity of FRET sensors is a major challenge that prevents their application in various fields and makes the use of expensive, high-end fluorometers necessary. Previously, we built a cost-effective, high-performance FRET analyzer that can specifically measure the ratio of two emission wavelength bands (530 and 480 nm) to achieve high detection sensitivity. More recently, it was discovered that FRET sensors with bacterial periplasmic binding proteins detect ligands with maximum sensitivity in the critical temperature range of 50 - 55 &#176;C. This report describes a protocol for assessing sugar content in commercially-available beverage samples using our portable FRET analyzer with a temperature-specific FRET sensor. Our results showed that the additional preheating process of the FRET sensor significantly increases the FRET ratio signal, to enable more accurate measurement of sugar content. The custom-made FRET analyzer and sensor were successfully applied to quantify the sugar content in three types of commercial beverages. We anticipate that further size reduction and performance enhancement of the equipment will facilitate the use of hand-held analyzers in environments where high-end equipment is not available.

This protocol describes the rapid and highly sensitive quantification of F&#246;rster resonance energy transfer (FRET) sensor data using a custom-made portable FRET analyzer. The device was used to detect maltose within a critical temperature range that maximizes detection sensitivity, enabling practical and efficient assessment of sugar content.

Recent improvements in F&#246;rster resonance energy transfer (FRET) sensors have enabled their use to detect various small molecules including ions and ...

Sheathless Capillary Electrophoresis&#8211;Mass Spectrometry for Metabolic Profiling of Biological Samples

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a protocol is presented for the analysis of anionic and cationic metabolites in biological samples by capillary electrophoresis&#8211;mass spectrometry (CE-MS). CE is well-suited for the analysis of highly polar and charged metabolites as compounds are separated on the basis of their charge-to-size ratio. A recently developed sheathless interfacing design, i.e., a porous tip interface, is used for coupling CE to electrospray ionization (ESI) MS. This interfacing approach allows the effective use of the intrinsically low-flow property of CE in combination with MS, resulting in nanomolar detection limits for a broad range of polar metabolite classes. The protocol presented here is based on employing a bare fused-silica capillary with a porous tip emitter at low-pH separation conditions for the analysis of a broad array of metabolite classes in biological samples. It is demonstrated that the same sheathless CE-MS method can be used for the profiling of cationic metabolites, including amino acids, nucleosides and small peptides, or anionic metabolites, including sugar phosphates, nucleotides and organic acids, by only switching the MS detection and separation voltage polarity. Highly information-rich metabolic profiles in various biological samples, such as urine, cerebrospinal fluid and extracts of the glioblastoma cell line, can be obtained by this protocol in less than 1 hr of CE-MS analysis.

A protocol for metabolic profiling of biological samples by capillary electrophoresis&#8211;mass spectrometry using a sheathless porous tip interface design is presented.

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a ...

Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find neurodegenerative diseases such as Alzheimer&#39;s and Parkinson&#39;s disease afflicting primarily the central nervous system, and systemic amyloidosis where serum amyloid A, transthyretin and IgG light chains deposit as amyloid in liver, carpal tunnel, spleen, kidney, heart, and other peripheral tissues. Amyloid has been known and studied for more than a century, often using amyloid specific dyes such as Congo red and Thioflavin T (ThT) or Thioflavin (ThS). In this paper, we present heptamer-formyl thiophene acetic acid (hFTAA) as an example of recently developed complements to these dyes called luminescent conjugated oligothiophenes (LCOs). hFTAA is easy to use and is compatible with co-staining in immunofluorescence or with other cellular markers. Extensive research has proven that hFTAA detects a wider range of disease associated protein aggregates than conventional amyloid dyes. In addition, hFTAA can also be applied for optical assignment of distinct aggregated morphotypes to allow studies of amyloid fibril polymorphism. While the imaging methodology applied is optional, we here demonstrate hyperspectral imaging (HIS), laser scanning confocal microscopy and fluorescence lifetime imaging (FLIM). These examples show some of the imaging techniques where LCOs can be used as tools to gain more detailed knowledge of the formation and structural properties of amyloids. An important limitation to the technique is, as for all conventional optical microscopy techniques, the requirement for microscopic size of aggregates to allow detection. Furthermore, the aggregate should comprise a repetitive &#946;-sheet structure to allow for hFTAA binding. Excessive fixation and/or epitope exposure that modify the aggregate structure or conformation can render poor hFTAA binding and hence pose limitations to accurate imaging.

Amyloid deposition is a hallmark of different diseases and afflicts many different organs. This paper describes the application of luminescent conjugated oligothiophene fluorescence staining in combination with fluorescence microscopy techniques. This staining method represents a powerful tool for detection and exploration of protein aggregates in both clinical and scientific setups.

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find ...

Dual DNA Rulers to Study the Mechanism of Ribosome Translocation with Single-Nucleotide Resolution

The ribosome translocation refers to the ribosomal movement on the mRNA by exactly three nucleotides (nt), which is the central step in protein synthesis. To investigate its mechanism, there are two essential technical requirements. First is single-nt resolution that can resolve normal translocation from frameshifting, during which the ribosome moves by other than 3 nt. The second is the capability to probe both the entrance and exit sides of mRNA in order to elucidate the whole picture of translocation. We report the dual DNA ruler assay that is based on the critical dissociation forces of DNA-mRNA duplexes, obtained by force-induced remnant magnetization spectroscopy (FIRMS).&#160; With 2-4 pN force resolution, the dual ruler assay is sufficient to distinguish different translocation steps. By implementing a long linker on the probing DNAs, they can reach the mRNA on the opposite side of the ribosome, so that the mRNA position can be determined for both sides. Therefore, the dual ruler assay is uniquely suited to investigate the ribosome translocation, and nucleic acid motion in general. We show representative results which indicated a looped mRNA conformation and resolved normal translocation from frameshifting.

Dual DNA ruler assay is developed to determine the mRNA position during ribosome translocation, which relies on the dissociation forces of the formed DNA-mRNA duplexes. With single-nucleotide resolution and capability of reaching both ends of mRNA, it can provide mechanistic insights for ribosome translocation and probe other nucleic acid displacements.

The ribosome translocation refers to the ribosomal movement on the mRNA by exactly three nucleotides (nt), which is the central step in protein synthesis. ...

Characterizing Cellular Proteins with In-cell Fast Photochemical Oxidation of Proteins

Fast photochemical oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting method used to characterize protein structure and interactions. FPOP uses a 248 nm excimer laser to photolyze hydrogen peroxide producing hydroxyl radicals. These radicals oxidatively modify solvent exposed side chains of 19 of the 20 amino acids. Recently, this method has been used in live cells (IC-FPOP) to study protein interactions in their native environment. The study of proteins in cells accounts for intermolecular crowding and various protein interactions that are disrupted for in vitro studies. A custom single cell flow system was designed to reduce cell aggregation and clogging during IC-FPOP. This flow system focuses the cells past the excimer laser individually, thus ensuring consistent irradiation. By comparing the extent of oxidation produced from FPOP to the protein&#39;s solvent accessibility calculated from a crystal structure, IC-FPOP can accurately probe the solvent accessible side chains of proteins.

Here, we characterize protein structure and interaction sites in living cells using a protein footprinting technique termed in-cell fast photochemical oxidation of proteins (IC-FPOP).

Fast photochemical oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting method used to characterize protein structure and interactions. ...

Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for hundreds of individual restriction endonuclease (REase) molecules in one experiment. This assay does not require protein labeling and can be carried out with a single imaging channel. In addition, the results of multiple individual experiments can be pooled to generate well-populated dwell-time distributions. Analysis of the resulting dwell-time distributions can help elucidate the DNA cleavage mechanism by revealing the presence of kinetic steps that cannot be directly observed. Example data collected using this assay with the well-studied REase, EcoRV - a dimeric Type IIP restriction endonuclease that cleaves the palindromic sequence GAT&#8595;ATC (where &#8595; is the cut site) - are in agreement with prior studies. These results suggest that there are at least three steps in the pathway to DNA cleavage that is initiated by introducing magnesium after EcoRV binds DNA in its absence, with an average rate of 0.17 s-1 for each step.

Using quantum-dot-labeled DNA and total internal reflection fluorescence microscopy, we can investigate the reaction mechanism of restriction endonucleases while using unlabeled protein. This single-molecule technique allows for massively multiplexed observation of individual protein-DNA interactions, and data can be pooled to generate well-populated dwell-time distributions.

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for ...

Cryo-EM and Single-Particle Analysis with Scipion

Cryo-electron microscopy has become one of the most important tools in biological research to reveal the structural information of macromolecules at near-atomic resolution. In single-particle analysis, the vitrified sample is imaged by an electron beam and the detectors at the end of the microscope column produce movies of that sample. These movies contain thousands of images of identical particles in random orientations. The data need to go through an image processing workflow with multiple steps to obtain the final 3D reconstructed volume. The goal of the image processing workflow is to identify the acquisition parameters to be able to reconstruct the specimen under study. Scipion provides all the tools to create this workflow using several image processing packages in an integrative framework, also allowing the traceability of the results. In this article the whole image processing workflow in Scipion is presented and discussed with data coming from a real test case, giving all the details necessary to go from the movies obtained by the microscope to a high resolution final 3D reconstruction. Also, the power of using consensus tools that allow combining methods, and confirming results along every step of the workflow, improving the accuracy of the obtained results, is discussed.

Single-particle analysis in cryo-electron microscopy is one of the main techniques used to determine the structure of biological ensembles at high resolution. Scipion provides the tools to create the whole pipeline to process the information acquired by the microscope and achieve a 3D reconstruction of the biological specimen.

Cryo-electron microscopy has become one of the most important tools in biological research to reveal the structural information of macromolecules at ...

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) establish and maintain the DNA methylation pattern. Changes to the normal DNA methylation pattern are linked to cancer development and progression, making DNMTs potential cancer drug targets. Thus, identifying and characterizing novel small molecule inhibitors of these enzymes is of great importance. This paper presents a protocol that can be used to screen for DNA methyltransferase inhibitors. The continuous coupled kinetics assay allows for initial velocities of DNA methylation to be determined in the presence and absence of potential small molecule inhibitors. The assay uses the methyl-sensitive endonuclease Gla I to couple methylation of a hemimethylated DNA substrate to fluorescence generation.
This continuous assay allows for enzyme activity to be monitored in real time. Conducting the assay in small volumes in microtiter plates reduces the cost of reagents. Using this assay, a small example screen was conducted for inhibitors of DNMT1, the most abundant DNMT isozyme in humans. The highly substituted anthraquinone natural product, laccaic acid A, is a potent, DNA-competitive inhibitor of DNMT1. Here, we examine three potential small molecule inhibitors &#8212; anthraquinones or anthraquinone-like molecules with one to three substituents &#8212; at two concentrations to describe the assay protocol. Initial velocities are used to calculate the percent activity observed in the presence of each molecule. One of three compounds examined exhibits concentration-dependent inhibition of DNMT1 activity, indicating that it is a potential inhibitor of DNMT1.

DNA methyltransferases are potential cancer drug targets. Here, a protocol is presented to assess small molecules for DNA methyltransferase inhibition. This assay utilizes an endonuclease to couple DNA methylation to fluorescence generation and allows for enzyme activity to be monitored in real time.&#160;

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) ...

Preparation of Nucleosome Core Particles Complexed with DNA Repair Factors for Cryo-Electron Microscopy Structural Determination

DNA repair in the context of chromatin is poorly understood. Biochemical studies using nucleosome core particles, the fundamental repeating unit of chromatin, show most DNA repair enzymes remove DNA damage at reduced rates as compared to free DNA. The molecular details on how base excision repair (BER) enzymes recognize and remove DNA damage in nucleosomes have not been elucidated. However, biochemical BER data of nucleosomal substrates suggest the nucleosome presents different structural barriers dependent on the location of the DNA lesion and the enzyme. This indicates the mechanisms employed by these enzymes to remove DNA damage in free DNA may be different than those employed in nucleosomes. Given that the majority of genomic DNA is assembled into nucleosomes, structural information of these complexes is needed. To date, the scientific community lacks detailed protocols to perform technically feasible structural studies of these complexes. Here, we provide two methods to prepare a complex of two genetically fused BER enzymes (Polymerase &#946; and AP Endonuclease1) bound to a single-nucleotide gap near the entry-exit of the nucleosome for cryo-electron microscopy (cryo-EM) structural determination. Both methods of sample preparation are compatible for vitrifying quality grids via plunge freezing. This protocol can be used as a starting point to prepare other nucleosomal complexes with different BER factors, pioneer transcription factors, and chromatin-modifying enzymes.

This protocol outlines in detail the preparation of nucleosomal complexes using two methods of sample preparation for freezing TEM grids.

DNA repair in the context of chromatin is poorly understood. Biochemical studies using nucleosome core particles, the fundamental repeating unit of ...

Determining the Thermodynamic and Kinetic Association of a DNA Aptamer and Tetracycline Using Isothermal Titration Calorimetry

The determination of binding affinity and behavior between an aptamer and its target is the most crucial step in selecting and using an aptamer for application. Due to the drastic differences between the aptamer and small molecules, scientists need to put much effort into characterizing their binding properties. Isothermal Titration Calorimetry (ITC) is a powerful approach for this purpose. ITC goes beyond determining disassociation constants (Kd) and can provide the enthalpy changes and binding stoichiometry of the interaction between two molecules in the solution phase. This approach conducts continuous titration using label-free molecules and records released heat over time upon the binding events produced by each titration, so the process can sensitively measure the binding between macromolecules and their small targets. Herein, the article introduces a step-by-step procedure of the ITC measurement of a selected aptamer with a small target, tetracycline. This example proves the versatility of the technique and its potential for other applications.

The present protocol describes the use of Isothermal Titration Calorimetry (ITC) to analyze the association and dissociation kinetics of the binding between a DNA aptamer and tetracycline, including sample preparation, running standards and samples, and interpreting the resulting data.

The determination of binding affinity and behavior between an aptamer and its target is the most crucial step in selecting and using an aptamer for ...