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 does comet assay help in DNA damage detection?
The comet assay or single-cell gel (SCG) test is a microgel electrophoresis technique that measures DNA damage at the level of single cells. A small number of cells suspended in a thin agarose gel on a microscope slide is lysed, electrophoresed, and stained with a fluorescent DNA binding dye.
What does the comet assay detect?
Single-Cell Gel Electrophoresis (Comet) Assay
Comet assay is a microgel electrophoresis technique, which detects DNA damage and repair in individual cells. The assay measures DNA damage (i.e., strand breaks, DNA adducts, excision repair sites, and cross-links) at the single-cell level.
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.
What is pulsed field gel electrophoresis used for?
Pulsed-field gel electrophoresis (PFGE) is a laboratory technique used by scientists to produce a DNA fingerprint for a bacterial isolate.
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).
How do you check for DNA damage?
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.
What is tail length in 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 are 5 kinds of pulsed field gel electrophoresis?
Different types of PFGE units are currently available including contour-clamped homogeneous electric field (CHEF) [71,72], transverse alternating field electrophoresis (TAFE) [73] and its relative ST/RIDEtm (Stratagene), and rotating gel electrophoresis (RGE) [74,75], with CHEF being the most common one.
What is the principle of pulsed field gel electrophoresis?
Pulse Field Gel Electrophoresis (PFGE) is a powerful genotyping technique used for the separation of large DNA molecules (entire genomic DNA) after digesting it with unique restriction enzymes and applying to a gel matrix under the electric field that periodically changes direction.
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.
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.
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.
What does tail DNA mean?
In the comet structure the undamaged DNA nucleoid part is referred to as “head” and the trailing damaged DNA streak is referred to as “tail.” The percentage of DNA in the tail is directly proportional to the percentage of DNA damage that has occurred in a particular cell.
What is the principle of pulsed-field gel electrophoresis?
What are the differences between pulsed-field gel electrophoresis and agarose gel electrophoresis?
Electrophoresis: The larger pieces of DNA are subjected to pulse field gel electrophoresis by applying electric current and altering its direction at regular intervals (in contrast to the conventional agarose gel electrophoresis done to separate the smaller fragments where the current is applied in a single direction).
What are the differences between pulsed field gel electrophoresis and agarose gel electrophoresis?
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 you reverse DNA damage?
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).
What does 2% agarose gel mean?
The concentration is measured in weight of agarose over volume of buffer used (g/ml). For a standard agarose gel electrophoresis, a 0.8% gel gives good separation or resolution of large 5–10kb DNA fragments, while 2% gel gives good resolution for small 0.2–1kb fragments.