Hi Aziz,
I think you are referring to an in vitro system. There are various methods you can use, but it depends on your purpose.
Single Gel Electrophoresis (Comet Assay) is one simple and well recognized fluorescence method to detect DNA strand breaks (both single and double strand breaks). You can find a protocol online easily.
If you desire to examine DNA condensation and formation of apoptotic bodies, Hoechst staining would be useful. There you can stain your cells with the Hoechst dye and detect the DNA condensation via fluorescence microscope or flow cytometry.
Hope this would help you.
a procedure for the comet assay, a gel electrophoresis–based method that can be used to measure DNA damage in individual eukaryotic cells. It is relatively simple to perform and sensitive. Although most investigations make use of its ability to measure DNA single-strand breaks, modifications to the method allow detection of DNA double-strand breaks, cross-links, base damage and apoptotic nuclei. The limit of sensitivity is approximately 50 strand breaks per diploid mammalian cell. DNA damage and its repair in single-cell suspensions prepared from yeast, protozoa, plants, invertebrates and mammals can also be studied using this assay. Originally developed to measure variation in DNA damage and repair capacity within a population of mammalian cells, applications of the comet assay now range from human and sentinel animal biomonitoring to measurement of DNA damage in specific genomic sequences. This protocol can be completed in fewer than 24 h.
in shaa allah, I will study the effect of some anthelmintic drugs on the DNA of some trematodal worms, so I will trace all papers dealing with methods used for detection of DNA damage . thanks for all
Many thanks all dear professors for your kind answering for my question
Note that the micronucleus assay only works with dividing cells. A related method that looks for micronuclei and apoptotic cells in sectioned tissue containing dividing cells is the nuclear anomaly test. Sections should be stained with Feulgen/fast green.
Comet Assay is without doubt a very reliable method, but a very easy, cheap, fast and in-situ method to detect DNA damage, is micronucleus frequency assay. It can be realised with all nucleated cells, like erythrocytes, haemocytes, bucal cells, hepatocytes, etc. To measure the micronucleus frequency, a blood smear can be prepared and stained and then it can be evaluated by a optical microscope
See https://www.ncbi.nlm.nih.gov/pubmed/16603255
and
Wargovich MJ, Goldberg MT, Newmark HL and Bruce WR (1983) Nuclear aberrations as a short-term test for genotoxicity to the colon: evaluation of nineteen agents in mice. JNCI 71, 133‑137
for details of micronucleus/nuclear anomaly methods.
Dear Mohammad Aziz,
You need to know what exactly you want to detect.
(target is - animal-rat or mouse, tissue,..( if in vivo?), cell line (if in vitro))
OECD 474, OECD 475, OECD 489.(google it)
I hope this helps you decide with the biological model
Dear Mohammad Aziz,
Greetings!!
You can go for Comet Assay (Single cell gel electrophoresis).
All the very best!!
With Regards,
Samrat Das
As mentioned by others, depends what you mean by DNA damage and what cell type. The standard (alkaline comet assay) detects primarily DNA gaps that form primarily as a result in an initial step in DNA repair. It needs quite a lot of equipment and specialist knowledge so collaboration with an expert in the field would be the way to go if you choose that. The micronucleus/nuclear anomaly test that I mentioned earlier requires standard histology and a good microscope (e.g. with a flat field x40 oil-immersion objective).
it was said that micronucleus assay is used for detection of chromosomal aberrations
Single-cell gel electrophoresis assay (comet assay), a visual biochemical technique is used to detect various genotoxic damages in mammalian cells
Article Toxoplasma gondii infection can induce retinal DNA damage: A...
Conference Paper Toxoplasma gondii infection can induce retinal DNA damage: a...
dear professor Raymond Proudlock
it is said that you need 1000x magnification by light microscopy to detect MN ?
also, MN not detected in rat ?
what is your opinion?
thanks
Hello Mohammad. Rats are now the most widely used species for micronucleus testing in the case of pharmaceuticals etc (as opposed to environmental studies). See OECD guideline 474 for details.
http://www.oecd.org/env/test-no-474-mammalian-erythrocyte-micronucleus-test-9789264264762-en.htm
Micronuclei (as mentioned in this context) refers to chromosome fragments or whole chromosomes that lag behind at anaphase and condense in the cytoplasm to form small DNA-containing bodies. Since they tend to get left behind in the transition erythroblast to erythrocyte, they are most readily detected in newly formed erythrocytes (termed 'polychromatic erythrocytes' or 'immature erythrocytes') because these do not contain an obscuring main nucleus. In rodents, the newly formed erythrocytes can be identified (differentiated) by their high RNA content (e.g. with Giemsa or Acridine orange stain). In mice, these micronucleated cells are released from the bone marrow into the blood where they gradually loose their RNA but retain the micronuclei. In rats and some other species, these micronucleated cells are sticky and are rapidly filtered out of the circulation. Therefore, in mice, the micronucleus test examines bone marrow or blood while in rats, examination is generally restricted to bone marrow.
In other species, newly formed erythrocytes may have only a very low RNA content or (as in birds, fish and reptiles) they may retain the main micronucleus. Makato Hayashi is regarded as a (or the) world expert on the test see.
Article The micronucleus test—most widely used in vivo genotoxicity test—
In practice, micronuclei can be induced by genotoxins in any dividing cell type but the methodology, timing and staining methods depend on the cell type being examined. Micronucleated immature ethryocytes are especially sensitive since they are a cohort of cells that will have been dividing during recent acute exposure to the chemical of interest.
Be aware that micronucleus-like bodies can be caused by other mechanisms (particularly incomplete enucleation in the case of erythrocytes or nuclear anomalies in the case of other cell types) so you should follow an established method appropriate for the species and cell-type to avoid complications. A DNA-specific stain is best (e.g. acridine orange); this gives excellent differentiation and also avoids problems with mast cell granules, which are problematic in rat bone marrow since they stick to erythrocytes and appear identical to micronuclei when rat bone marrow smears are stained with Giemsa.
Before you embark on any practical testing, I'd suggest a literature search to look for authors with relevant practical experience then you can politely request their review of your proposal. That will save a lot of work and help avoid necessary or inappropriate experimentation on animals.
Micronuclei are best observed at about 400 to 630 times magnification using a plan oil-immersion objective - least tiring and best optics. 1000 x is OK but scanning will be tedious. You will need a fluorescence microscope if you decide to use acridine orange stain. For non-erythrocytes, Feulgen/light (or pale) green is also useful and very specific.
In summary, micronucleus testing uses very little equipment and is very simple but, like all genetox tests, you need to know what you are doing to make the effort worthwhile.
COMET Assay, Immunostaining with DNA damage markers like 53BP1 or gH2AX.
