Name: Dot Blot Assay to Quantify Viral Genome
Uploaded: 2023-06-29T19:11:38
Description: 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 ...

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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 mak...

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.

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Biological Techniques

Blot Techniques

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

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