What is tail moment comet assay?
The tail moment is defined as the product of the tail length and the fraction of total DNA in the tail (Tail moment=tail length x % of DNA in the tail). This is calculated automatically by the computer software system as an average for the 50 cells selected for measurement.
What is Olive moment in comet assay?
The tail moment calculated by Olive et al. (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.
Why is tail DNA recommended above mean tail moment for reporting DNA damage?
With respect to providing smaller variability in quantiles for the amount of DNA damage, however, the tail moment is the preferred parameter for both groups. Moreover, the tail moment provides the most stable estimates for DNA damage because it has a larger degree of uniformity in quantile dispersions.
What is tail DNA?
In the comet structure, the undamaged DNA nucleoid part is referred to as the “head” and the trailing damaged DNA streak is referred to as the “tail” (Figure 43.4). The percentage of DNA in the tail is directly proportional to the percentage of DNA damage that has occurred in a particular cell.
How can you detect DNA damage?
DNA breaks and lesions may be detected by PCR or using agarose gel electrophoresis (7). PCR is one of the most frequently used techniques for detecting DNA damage (7).
Is comet assay quantitative?
Combining single cell electrophoresis, fluorescent microscopy, and image interpretation, both the alkaline and neutral comet assays provide quantitative approaches to evaluate the extent of DNA damage in vitro.
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.
What is alkaline comet assay?
The alkaline comet assay is a sensitive and relatively inexpensive technique for the detection of DNA damage and DNA repair (1).
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.
How is DNA repair measured?
Generally, there are two ways to measure repair rates. one is to monitor the removal of the DnA damage; the other is to monitor the restoration of the activity of the damaged DnA. techniques have been developed to monitor removal of specific lesions such as pyrimidine dimers by using T4 endonuclease V.
How do you repair damaged DNA?
Most damage to DNA is repaired by removal of the damaged bases followed by resynthesis of the excised region. Some lesions in DNA, however, can be repaired by direct reversal of the damage, which may be a more efficient way of dealing with specific types of DNA damage that occur frequently.
How can you quantify DNA damage?
Quantitative PCR (qPCR) has been performed to quantify the amount of DNA damage on both strands, as well as the kinetics of DNA damage removal in the mitochondrial DNA (mtDNA) of human and other organisms (7,9).
What is comet assay positive control?
The positive control should be an established genotoxic substance known to induce DNA damage detectable by the Comet assay. The positive control dose(s) should be chosen such that a positive response is obtained but does not immediately reveal the identity of the coded slides to the scorer.
What temperature does low melt agarose melt?
This low melting temperature (65º C) agarose is ideally suited for direct enzymatic manipulation of nucleic acids in remelted agarose without additional…
Why is it better to use low melting point agarose for the separation of DNA from agarose?
DNA of a given size runs slightly faster through gels cast with low-melting-temperature agarose than through conventional agarose gels. For this reason, the voltage applied to low-melting-temperature agarose gels should be lower than that applied to standard agarose gels.
How do you detect DNA damage response?
How do you measure base excision repair?
Base excision repair or NER can be measured by using substrate nucleoids with appropriate DNA lesions.
What vitamin helps with DNA repair?
Vitamin C supplementation was potentially beneficial, because an increase in DNA repair incision capacity was observed, which was not seen in well-nourished subjects.
Can DNA damage be reversed?
Double-strand breaks, the most serious injuries that happen to DNA, can be repaired by one of two pathways: a fast but error-prone process known as NHEJ (non-homologous end joining) and a slower, error-free pathway known as HR (homologous recombination).
Why do we use low melting agarose?
You use low melting point agar as it melts near a physiological temperature that will not denature proteins, nucleic acids and such.
Why is low melting agarose used?
Agarose II (Low Melting Agarose)
Agarose II has a low melting and gelling temperature compared to standard agaroses. The low melting temperature allows for the recovery of undamaged nucleic acids below the denaturation temperature.
Why do we use 1.5% agarose gel?
High percentage agarose gels (e.g. 1.5%) are used for the separation of small DNA molecules (100 – 1000 base-pairs in length), while low percentage gels (e.g. 0.6%) are used for large molecules (104 – 105 base-pairs).
What is the difference between normal agarose and low melting agarose?
LM agaroses have lower gel strength than standard agaroses, yet they can be handled easily. LM agaroses have higher clarity (gel transparency) than gels of standard agaroses. LM agaroses have great sieving capacity. The gelling temperature of LM agaroses is 24 to 28°C.
How many DNA breaks a day?
Humans, per cell per day: 10,000. 11,500. 2,800 specific damages 8-oxoGua, 8-oxodG plus 5-HMUra.
What are three ways that DNA gets damaged?
Endogenous sources of DNA damage include hydrolysis, oxidation, alkylation, and mismatch of DNA bases; sources for exogenous DNA damage include ionizing radiation (IR), ultraviolet (UV) radiation, and various chemicals agents.