Monday, June 07, 2004
The Comet Assay for detection of DNA repair and DNA damage by tail intensity. In Comet Assays the damage is detected with the single-stranded or double-stranded DNA breaks. DNA damage can be induced by chemicals or UV light radiation. A single cell is paced in low melting agarose on a microscope slide. The cell membrane will be lysed with detergent and salt concentration. Nucleotides will be released from the nucleus and electrophoresis will migrate the damaged DNA to the + anode electric field. The more the DNA is damaged the more there will appear a migration tail. Endonucleases damage increases the DNA migration, whereas DNA-DNA and DNA-protein cross-links result in retarded DNA migration, Tice, 2000. The comet assay is used in DNA repair studies, in animal and clinical studies for base excision repair (BER), nucleotide excision repair (NER) like the Vitotox L. Gevaert, 2009 measures the cytotoxicity in Salmonella lux promotor cloned cells. They show that the Comet Assay or single cell gel electrophoresis (SCGE) assay is consistent detection method for DNA damage, Singh, 1988. The Comet assays are measuring DNA breaks and loss of supercoiling from oxidative stress. For measuring DNA damage from apoptosis, cell death by cytotoxicity and strand breaks at incomplete excision repair sites or alkali-labile sites about 6500 papers have been published in PubMed and identified by GENTAUR.
For the comet assay procedure GENTAUR also supplies the Comet microscope glass slides. Cellular repair resolves the DNA damage and nucleoids containing specific DNA and the breaks are tested with the comet assay. Performed on Drosophila melanogaster cells or for human DNA damage studies for cancer susceptibility by environmental factors, food habits, sport. A fluorescence microscopy is needed to observe the comet tail where the intensity, length and brightness of the tail indicates the number of DNA lesions and DNA unwinding at neutral pH 7.5. At pH 12.3 the comet assay detects single and double strand breaks, incomplete excision repair sites and cross linking. The DNA amount from the cell nucleus is an indicator of the incurred DNA damage. High throughput is achieved with the 96-Well CometChip the fastest system for high-throughput measurement of DNA damage. Alkali labile sites (ALS) are susceptible of DNA breaching, Miyamae, 1997 and will fast migrate out of the cell from DNA damage, Fairbairn, 1995. The Comet Assay can be applied to both eukaryotic chromosomes and prokaryotic cells. For oncology testing of invasive cancer in the human body the Comet is used by B Vilhar
Isotype or positive controls by peptides, antibodies and deactivated samples.
Horizontal protein electrophoresis in PAGE gels or polyacrylamide gels and vertical DNA electrophoresis is performed in agarose. GENTAUR also supplies the buffers.