Increasing the resolution of the comet assay using fluorescent in situ hybridization--a review.
The comet assay (single-cell gel electrophoresis) is now the most popular method for measuring low levels of damage in cellular DNA. Cells are embedded in agarose on a microscope slide and lysed to produce nucleoids of supercoiled DNA attached to the nuclear matrix. Breaks in the DNA relax the supercoiling and allow DNA loops to expand, and on electrophoresis to move towards the anode, giving the appearance of a comet tail. The % of DNA in the tail reflects the break frequency. Digestion of nucleoid DNA with lesion-specific endonucleases extends the usefulness of the method to investigate different kinds of damage. DNA repair can be studied by treating cells with a genotoxic agent, incubating them and using the comet assay to follow the removal of the damage. An important feature of the assay is that damage is detected at the level of individual cells. The comet assay can be combined with fluorescent in situ hybridization, using labelled probes to particular DNA sequences, and DNA damage and repair can be examined at an even finer level of resolution. Here, we provide a general review of the technique, answer some technical and theoretical questions and give examples of applications of the method.
Shaposhnikov
Sergey
S
Department of Nutrition, Institute of Basic Medical Sciences, University of Oslo, PB 1046 Blindern, 0316 Oslo, Norway. sergey.shaposhnikov@medisin.uio.no
Frengen
Eirik
E
Collins
Andrew R
AR
D002843:Chromatin / Q000737:chemistry; D020552:Comet Assay / Q000379:methods*; D015342:DNA Probes / Q000378:metabolism; D004260:DNA Repair; D004194:Disease / Q000235:genetics; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*
D002843:Chromatin; D015342:DNA Probes