What are the limitations of comet assay?
Table of Contents
What are the limitations of comet assay?
2.5.6 Pros and cons
| Pros | Cons |
|---|---|
| Comet assay is easy, sensitive, and more accurate in the determination of DNA damage and cell death | Comet assay is tedious and can damage the membrane of cells altering the distribution of live, apoptotic, and/or necrotic cell population |
How do you do a comet assay?
Comet assay utilizes single cells to measure DNA damage. First, cells are embedded into agarose and then placed onto a slide. The slide is then immersed into lysis solution to break open the cell membrane. After the cells are lysed, DNA is denatured under neutral or alkaline conditions and run through electrophoresis.
What does the comet assay measure?
The comet assay (single-cell gel electrophoresis) is a simple method for measuring deoxyribonucleic acid (DNA) strand breaks in eukaryotic cells. Cells embedded in agarose on a microscope slide are lysed with detergent and high salt to form nucleoids containing supercoiled loops of DNA linked to the nuclear matrix.
What can comet assay study?
Comet Assay is a valuable tool in measuring the extent of DNA damage and its quantification in populations exposed to different types of genotoxic agents and environmental pollutants.
How many cells does a comet assay have?
Thus by counting a representative sample of ∼ 100–300 cells per tissue it is possible to arrive at the average percentage of DNA damage accumulated in a particular tissue due to genotoxic stress. The comet assay is also referred to as SCGE assay.
Why low melting agar is used in comet assay?
Low melting agarose is melts at low temperature with fine quality powder and higher price incomarision with normal agarose Low melting agarose is very much suitable for comet assay. You can use also Normal agarose also.
What is tail intensity in comet assay?
Electrophoresis at high pH results in structures resembling comets, observed by fluorescence microscopy; the intensity of the comet tail relative to the head reflects the number of DNA breaks. The likely basis for this is that loops containing a break lose their supercoiling and become free to extend toward the anode.
What is Olive tail moment in comet assay?
(1990) came to be known as the Olive tail moment (OTM). This parameter is considered to be particularly useful in describing heterogeneity within a cell population, as OTM can pick up variations in DNA distribution within the tail. Table 1 List of various Comet Assay parameters used in published literature.
How does voltage affect gel electrophoresis?
The higher the voltage, the faster the DNA will travel through the gel. However, voltages that are too high can possibly melt the gel or cause smearing or distortion of DNA bands. The gel concentration and volume (thickness) affect electrophoretic separation.
When performing gel electrophoresis How could you determine the size of the DNA fragments?
Using electrophoresis, we can see how many different DNA fragments are present in a sample and how large they are relative to one another. We can also determine the absolute size of a piece of DNA by examining it next to a standard “yardstick” made up of DNA fragments of known sizes.
What is neutral comet assay?
Comet assay under neutral conditions allows the detection of DNA double-strand breaks (DSB), considered to be the biologically relevant radiation-induced lesion.
What does the electrophoresis apparatus consist of?
The gel electrophoresis apparatus consists of a gel, which is often made from agar or polyacrylamide, and an electrophoretic chamber (typically a hard plastic box or tank) with a cathode (negative terminal) at one end and an anode (positive terminal) at the opposite end.
What size DNA fragments will travel the farthest?
shortest
Because DNA is negatively-charged, it moves toward the positive electrode. The DNA fragments that are shortest will travel farthest, while the longest fragments will remain closest to the origin.
What does using a higher voltage in the electrophoresis apparatus change about the experiment?
What affects the resolution of gel electrophoresis?
The concentration of gel affects the resolution of DNA separation. The agarose gel is composed of microscopic pores through which the molecules travel, and there is an inverse relationship between the pore size of the agarose gel and the concentration – pore size decreases as the density of agarose fibers increases.
What is resolution gel electrophoresis?
The resolution of agarose gel electrophoresis for DNA separation is mainly dominated by the concentration of agarose gel and working voltage of electrophoresis. In most cases, dispersed and tailed DNA bands were obtained after electrophoresis, accompanying with serious background signals derived from EB dye.
