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NOTE: Recipes for the solutions and buffers needed for this protocol are provided in&#160;Table 1.
1. Dot blot
<ol>
	<li>Preparation of plasmid DNA standards
	<ol>
		<li>Dilute AAV vector genome plasmid DNA to 10 ng/&#181;L in water or Tris-HCl buffer (10 mM, pH 8.0). Take 25 &#181;L of this dilution and digest with an appropriate restriction enzyme for 1 h to linearize the plasmid DNA in a reaction volume of 50 &#181;L. 
		NOTE: We make a duplicated set of digestion (see step 1.4.1). The appropriate enzyme should be one that cuts the plasmid DNA outside the dot blot probe-binding region. For convenience, the diluted plasmid DNA can be aliquoted (25 &#181;L/tube) and stored frozen at -20 &#176;C for future use. Digest the plasmid DNA while the tubes are shaking in step 5.1. Do not over-digest the plasmid DNA standard.</li>
		<li>Add 450 &#181;L of water or TE to the tube containing the digested plasmid DNA standard and mix thoroughly. Transfer 70 &#181;L of this mixture to a new 1.5 mL microcentrifuge tube with 1,330 &#181;L of water or TE to make a diluted plasmid standard (25 pg/&#181;L).</li>
		<li>Follow&#160;Table 1&#160;to create a set of two-fold serial dilutions (600 &#181;L/tube). Mix the dilutions thoroughly by vortexing for 5 s.</li>
	</ol>
	</li>
	<li>Denaturing of standards and viral DNA samples
	<ol>
		<li>Add 600 &#181;L of 2x Alkaline Solution to each standard dilution. Mix well by vortexing for 5 to 10 s. Incubate at room temperature for 10 min.</li>
		<li>Add 120 &#181;L of 2x Alkaline Solution to each viral DNA sample. Mix well by vortexing for 5 to 10 s. Incubate at room temperature for 10 min.</li>
	</ol>
	</li>
	<li>Setting up the dot blot apparatus
	<ol>
		<li>Using scissors, cut a blotting (e.g., zeta-probe) membrane to an appropriate size for the number of samples and standards. Soak the membrane with water for 10 min before placing it on a dot blot apparatus. Cover unused wells on the membrane apparatus. 
		NOTE: Handle the membrane with clean tweezers. To cover empty wells, the light blue protection sheet that comes with the membrane can be used. Do not allow the membrane to dry prior to binding samples and standards. For more details on the assembly and use of the apparatus, please refer to the user manual.</li>
		<li>Add water to the wells to which samples will be loaded. Apply vacuum and pull water through the wells to check for errors and retest when fixed. Re-tighten the screws while applying vacuum to ensure tight sealing. 
		NOTE: Incomplete sealing may cause sample leakage between the wells.</li>
		<li>Once water is completely pulled through, release the vacuum completely (i.e., the vacuum manifold should be open to air pressure).</li>
	</ol>
	</li>
	<li>Loading standards and samples to the dot blot apparatus
	<ol>
		<li>Apply 400 &#181;L of each diluted plasmid DNA standard to each well, and run four lanes of standard dilutions. Use two separate aliquots of the standard digest and load each in duplicate. Apply 200 &#181;L/well of each viral DNA sample. 
		NOTE: Using the remaining ~40 &#181;L of denatured samples, diluted samples can be prepared (e.g., 10-fold diluted samples using 20 &#181;L of the sample plus 180 &#181;L of 1x Alkaline Solution) and blotted if necessary.</li>
		<li>Apply a gentle vacuum to pull the DNA solutions through the well. 
		NOTE: Vacuum pressure needs to be adjusted by partially opening a three-way valve so that the vacuum pressure is applied to the dot blot apparatus as well as the atmosphere (i.e., with the stopcock arms positioned at an approximately 45&#176; angle where it makes a louder suction noise).</li>
		<li>Once all the wells have emptied, release the vacuum by adjusting the three-way valve. Add 400 &#181;L of 1x Alkaline Solution to each well that contained standards and samples. Wait for 5 min before re-applying the vacuum to empty the wells.</li>
		<li>Re-apply the vacuum in the same way.</li>
		<li>Disassemble the dot blot apparatus under the vacuum, remove the membrane, and rinse it with 2x SSC. Place the membrane on a clean paper towel with the DNA-bound side facing up to remove excess 2x SSC.</li>
		<li>UV-crosslink the blotted DNA to the membrane with an appropriate UV crosslinker; the membrane is now ready for hybridization. 
		NOTE: Wet membranes can be used for UV crosslinking. The dried, UV-crosslinked membranes can be stored at room temperature. Further information can be found in the&#160;Table of Materials.</li>
	</ol>
	</li>
</ol>
2. Hybridization and washing
<ol>
	<li>Warm up the Hybridization Buffer in a 65 &#176;C water bath.</li>
	<li>Enzymatically label a DNA probe with radioactive &#945;-32P dCTP and purify it using commercially available kits according to the manufacturer&#39;s recommendation. 
	NOTE: We use a probe of 0.5&#8211;2.0 kb in length from an enhancer-promoter region or a protein-coding sequence in the viral genome. Although this protocol utilizes&#160;32P-labeled radioactive probes for signal detection, non-radioactive chemiluminescent or fluorescent probes can also be used (please refer to the&#160;Discussion&#160;section).</li>
	<li>Place the membrane in a hybridization bottle with the DNA-bound side up, rinse the membrane with 5 mL prewarmed Hybridization Buffer, and discard the buffer. Then add 10 mL of prewarmed Hybridization Buffer and place the bottle in a rotating hybridization oven set at 65 &#176;C. Rotate for &#8805;5 min.</li>
	<li>Heat-denature 20 &#181;L of 10 mg/mL sheared herring or salmon sperm DNA solution and the&#160;32P-labeled probe (&#8805;107&#160;cpm) for 5 min by placing the tubes on a heat block set at 100 &#176;C. Then snap-chill them on ice for &#8805;2 min, spin briefly, and keep them on ice until used. 
	Caution: For radioactive DNA probes, 1.5 mL tubes with screw caps and O-rings must be used.</li>
	<li>Quickly add the denatured sperm DNA and radioactive probe to the Hybridization Buffer in the hybridization bottle and shake the bottle for 10 s to mix. Return the bottle to the 65 &#176;C oven and incubate the bottle with rotation at 65 &#176;C for &#8805;4 h.</li>
	<li>Once hybridization is complete, stop the rotation, remove the hybridization bottle, and then pour the radioactive probe solution into a 50 mL conical tube with a leak-proof plug seal cap. Store the probe in an appropriate container placed in a refrigerator designated for radioactive materials. 
	NOTE: Used Hybridization Buffer with a probe that is stored at 4 &#176;C can be re-used at least 5 times by placing the 50 mL conical tube with a leak-proof plug seal cap that contains Hybridization Buffer in a 100 &#176;C water bath for 5 min.</li>
	<li>Wash the membrane with Wash Buffer heated to 65 &#176;C. Add 20 to 30 mL of Wash Buffer to the hybridization bottle and rotate for 5 min. Repeat this wash 2 more times. 
	NOTE: Measure the radioactivity of wash solutions and record it if required by the local institute.</li>
	<li>While washing the membrane, place a phosphor imaging screen on an image eraser for 5 min.</li>
	<li>After the third wash, remove the membrane from the hybridization bottle, remove the excess buffer on the membrane with paper towels, and place the membrane in a clear plastic paper holder. Check radioactive signals on the membrane using a Geiger counter. Expose the erased phosphor imaging screen to the membrane for 10 min to overnight, depending on the signal intensity.</li>
	<li>Scan the screen using a phosphor image scanning system and obtain the data on the signal intensity of each dot.</li>
</ol>
Table 1: Quantitative dot blot plasmid DNA standards.&#160;Necessary volumes of 25 pg/&#181;L plasmid DNA standard solution and water or TE to prepare plasmid DNA standards by two-fold serial dilutions (600 &#181;L/tube) are shown. The numbers in the plasmid DNA standard column (0.039 to 5 ng) are the quantity of DNA in 200 &#181;L of each plasmid DNA standard solution.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Plasmid DNA standard (ng)</td>
			<td>Volumes (&#181;L) of 25 pg/&#181;L plasmid DNA&#160; standard solution</td>
			<td>Volumes (&#181;L) of water</td>
			<td>Total volume (&#181;L)</td>
		</tr>
		<tr>
			<td>5</td>
			<td>600</td>
			<td>0</td>
			<td>600</td>
		</tr>
		<tr>
			<td>2.5</td>
			<td>300</td>
			<td>300</td>
			<td>600</td>
		</tr>
		<tr>
			<td>1.25</td>
			<td>150</td>
			<td>450</td>
			<td>600</td>
		</tr>
		<tr>
			<td>0.625</td>
			<td>75</td>
			<td>525</td>
			<td>600</td>
		</tr>
		<tr>
			<td>0.313</td>
			<td>37.5</td>
			<td>562.5</td>
			<td>600</td>
		</tr>
		<tr>
			<td>0.156</td>
			<td>18.8</td>
			<td>581.2</td>
			<td>600</td>
		</tr>
		<tr>
			<td>0.078</td>
			<td>9.4</td>
			<td>590.6</td>
			<td>600</td>
		</tr>
		<tr>
			<td>0.039</td>
			<td>4.7</td>
			<td>595.3</td>
			<td>600</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Benzonase</td>
			<td>MilliporeSigma</td>
			<td>1016970001</td>
			<td>Referred to as&#160;Serratia marcescen endonuclease&#160;in the main text.</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Roche</td>
			<td>4716728001</td>
			<td>Referred to as DNase I Enzyme A in the main text.</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Invitrogen</td>
			<td>18047019</td>
			<td>Referred to as DNase I Enzyme B in the main text.</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>New England Biolabs</td>
			<td>M0303L</td>
			<td>Referred to as DNase I Enzyme C in the main text.</td>
		</tr>
		<tr>
			<td>1.5 mL Attached O-Ring Screw Cap Microcentrifuge Tubes</td>
			<td>Corning</td>
			<td>430909</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Polypropylene Conical Tube</td>
			<td>Corning</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr>
			<td>3 M Sodium Acetate</td>
			<td>Teknova</td>
			<td>S0298</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Polypropylene Conical Tube</td>
			<td>Corning</td>
			<td>430291</td>
			<td></td>
		</tr>
		<tr>
			<td>AccuGENE 0.5 M EDTA Solution</td>
			<td>Lonza</td>
			<td>51234</td>
			<td></td>
		</tr>
		<tr>
			<td>AccuGENE 1 M Tris-HCl pH 8.0</td>
			<td>Lonza</td>
			<td>51238</td>
			<td></td>
		</tr>
		<tr>
			<td>AccuGENE 10% SDS</td>
			<td>Lonza</td>
			<td>51213</td>
			<td></td>
		</tr>
		<tr>
			<td>AccuGENE 1X TE Buffer</td>
			<td>Lonza</td>
			<td>51235</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Dot Apparatus</td>
			<td>Bio-Rad</td>
			<td>1706545</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>MilliporeSigma</td>
			<td>A3294</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Lifter</td>
			<td>Corning</td>
			<td>3008</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>MilliporeSigma</td>
			<td>372978</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA Extractor Kit</td>
			<td>Wako Pure Chemical Industries</td>
			<td>295-50201</td>
			<td>This is used for quantitative dot blots on purified virus. We use only a half volume of all the reagents in each step recommended for the Protocol Scheme 2 in the manual provided by the manufacturer.</td>
		</tr>
		<tr>
			<td>Ethanol 200 Proof</td>
			<td>Decon Labs</td>
			<td>2716</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll 400</td>
			<td>Alfa Aesar</td>
			<td>B22095</td>
			<td>Referred to as Polysucrose 400.</td>
		</tr>
		<tr>
			<td>Glycogen</td>
			<td>Roche</td>
			<td>10901393001</td>
			<td></td>
		</tr>
		<tr>
			<td>Herring Sperm DNA</td>
			<td>Invitrogen</td>
			<td>15634-017</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization Tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>13-247-150</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageQuant TL</td>
			<td>GE Healthcare Life Sciences</td>
			<td>ImageQuant TL</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Choloride Hexahydrate</td>
			<td>MilliporeSigma</td>
			<td>M0250</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphorimaging Exposure Cassette</td>
			<td>GE Healthcare Life Sciences</td>
			<td>Various</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphorimaging Screen</td>
			<td>GE Healthcare Life Sciences</td>
			<td>Various</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid Maxi Kit</td>
			<td>QIAGEN</td>
			<td>12162</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylenimine</td>
			<td>Polysciences Inc</td>
			<td>23966-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyvinylpyrrolidone</td>
			<td>MilliporeSigma</td>
			<td>P5288</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K Solution</td>
			<td>Invitrogen</td>
			<td>25530-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Restriction Enzymes</td>
			<td>New England Biolabs</td>
			<td>Various</td>
			<td></td>
		</tr>
		<tr>
			<td>Salmon Sperm DNA</td>
			<td>Invitrogen</td>
			<td>15632011</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological Pipettes</td>
			<td>Thermo Fisher Scientific</td>
			<td>13-678-11D / 13-678-11E / 13-678-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Thermo Fisher Scientific</td>
			<td>S271-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Citrate Tribasic Dihydrate</td>
			<td>MilliporeSigma</td>
			<td>C8532</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP152-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Typhoon FLA 7000</td>
			<td>GE Healthcare Life Sciences</td>
			<td>28955809</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure Buffer-Saturated Phenol</td>
			<td>Invitrogen</td>
			<td>15513-047</td>
			<td></td>
		</tr>
		<tr>
			<td>UV Crosslinker</td>
			<td>Spectroline</td>
			<td>XLE-1000</td>
			<td>Optimal Crosslink mode is used, providing a UV energy dosage of 120 mJ/cm2.</td>
		</tr>
		<tr>
			<td>Zeta-Probe Membrane</td>
			<td>Bio-Rad</td>
			<td>162-0165</td>
			<td></td>
		</tr>
	</tbody>
</table>

dot blot assay to quantify viral genome

Source: Powers, J. M. et al., <a href="https://app.jove.com/v/56766">A Quantitative Dot Blot Assay for AAV Titration and Its Use for Functional Assessment of the Adeno-associated Virus Assembly-activating Proteins.</a>&#160;J. Vis. Exp. (2018).
In this video, we describe the dot blot assay to quantify a viral genome. The signal intensity is directly related to the amount of viral DNA in the sample.

Dot Blot Assay to Quantify Viral Genome

Source: Powers, J. M. et al., A Quantitative Dot Blot Assay for AAV Titration and Its Use for Functional Assessment of the Adeno-associated Virus ...

To quantify viral DNA using the dot blot assay, begin with an isolated viral DNA suspension. Treat with an alkaline solution to denature the double-stranded DNA into single-strands for subsequent hybridization.
Assemble the dot blot apparatus with a pre-wetted nylon membrane. Load denatured viral DNA and linearized standard plasmid DNA solutions into the wells. Apply a suitable vacuum, facilitating effective negatively-charged viral single-stranded DNA transfer and binding onto the positively-charged membrane surface through electrostatic interactions, forming dot patterns.
Following the run, wash the membrane with sodium citrate buffer, helping improve the assay sensitivity. Post-drying, expose the membrane to ultraviolet light, crosslinking the blotted DNA to the membrane.
Add a heat-denatured, non-heterologous DNA fragment and viral genome-specific radioactive phosphorus-labeled DNA probe suspension in hybridization buffer to the membrane. Incubate, facilitating hybridization.
The DNA fragments act as blocking agents, binding to the remaining membrane regions, minimizing non-specific probe binding. The radioactive probe hybridizes with the complementary sequence on the viral single-stranded DNA.
Wash with wash buffer to remove unhybridized probes. Using the phosphor image scanning system, quantify the radioactive signals emitted by the radioactive probes hybridized to the viral genome on the membrane, to obtain dot blot signals.
From the standard curve, the signal intensity of each dot on the membrane can be used to calculate the amount of viral DNA in the sample.

Research

JoVE Encyclopedia of Experiments

Biological Techniques

Blot Techniques

537 Views. Source: Powers, J. M. et al., A Quantitative Dot Blot Assay for AAV Titration and Its Use for Functional Assessment of the Adeno-associated Virus Assembly-activating Proteins. J. Vis. Exp. (2018). In this video, we describe the dot blot assay to quantify a viral genome. The signal intensity is directly related to the amount of viral DNA in the sample. 

Video: Dot Blot Assay to Quantify Viral Genome - Concept

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global DNA methylation level of chondrocytes is considered to be a suitable biomarker for the loss of the chondrocyte phenotype. However, results based on different experimental methods can be inconsistent. Therefore, it is important to establish a precise, simple, and rapid method to quantify global DNA methylation levels during chondrocyte dedifferentiation.
Current genome-wide methylation analysis techniques largely rely on bisulfite genomic sequencing. Due to DNA degradation during bisulfite conversion, these methods typically require a large sample volume. Other methods used to quantify global DNA methylation levels include high-performance liquid chromatography (HPLC). However, HPLC requires complete digestion of genomic DNA. Additionally, the prohibitively high cost of HPLC instruments limits HPLC&#39;s wider application.
In this study, genomic DNA (gDNA) was extracted from human chondrocytes cultured with varying number of passages. The gDNA methylation level was detected using a methylation-specific dot blot assay. In this dot blot approach, a gDNA mixture containing the methylated DNA to be detected was spotted directly onto an N+ membrane as a dot inside a previously drawn circular template pattern. Compared with other gel electrophoresis-based blotting approaches and other complex blotting procedures, the dot blot method saves significant time. In addition, dot blots can detect overall DNA methylation level using a commercially available 5-mC antibody. We found that the DNA methylation level differed between the monolayer subcultures, and therefore could play a key role in chondrocyte dedifferentiation. The 5-mC dot blot is a reliable, simple, and rapid method to detect the general DNA methylation level to evaluate chondrocyte phenotype.

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

We present a method to quantify DNA methylation based on the 5-methylcytosine (5-mC) dot blot. We determined the 5-mC levels during chondrocyte dedifferentiation. This simple technique could be used to quickly determine the chondrocyte phenotype in ACI treatment.

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global ...

JoVE Journal

Developmental Biology

Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that may facilitate the development of vaccines and peptide drugs. Peptide scanning is a simple and efficient method that straightforwardly maps the linear epitope recognized by a monoclonal antibody (mAb). Here, the authors present an epitope determination methodology involving serially truncated recombinant proteins, synthetic peptide design, and dot-blot hybridization for the antigenic recognition of nervous necrosis virus coat protein using a neutralizing mAb. This technique relies on the dot-blot hybridization of synthetic peptides and mAbs on a polyvinylidene fluoride (PVDF) membrane. The minimum antigenic region of a viral coat protein recognized by the RG-M56 mAb can be narrowed down by step-by-step trimmed peptide mapping onto a 6-mer peptide epitope. In addition, alanine scanning mutagenesis and residue substitution can be performed to characterize the binding significance of each amino acid residue making up the epitope. The residues flanking the epitope site were found to play critical roles in peptide conformation regulation. The identified epitope peptide may be used to form crystals of epitope peptide-antibody complexes for an x-ray diffraction study and functional competition, or for therapeutics.

Here, the authors present a simple and efficient protocol to define a linear antigenic epitope using a purified monoclonal antibody and peptide scanning through dot-blot hybridization. The identified epitope can then be used in therapeutic and diagnostic applications.

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that ...

Immunology and Infection

The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic mechanisms have been intensively studied. 5-hydroxymethylcytosine displays dynamic features during embryonic and postnatal development of the brain, plays a regulatory function in gene expression, and is involved in multiple neurological disorders. Here, we describe the detailed methods including immunofluorescence staining and&#160;DNA dot-blot to detect 5-hydroxymethylcytosine in cultured cells and brain tissues of mouse.

Here, we present a protocol to detect 5-hydroxymethylcytosine in cells and brain tissues, utilizing immunofluorescence staining and DNA dot-blot methods.

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic ...

Dot Blot Assay to Quantify Viral Genome

Transcript