Comet Assay
A Complete Andor System Solution: Komet 5™ & Electron Multiplying CCD (EMCCD)
Comet Impact!
Comet assay? Not a technique in astronomy whereby the solar system and beyond is
scanned to assess the risk of the human race following the fate of the dinosaurs!
The Comet Assay, also known as "Single Cell Gel (SCG) electrophoresis", is a highly
sensitive and efficient means of assessing and quantifying DNA damage and repair
at the level of the individual cell, applicable to any eukaryotic cell. Types of
DNA damage include single-/double-strand breaks, DNA-drug crosslinking, DNA-protein
or DNA-DNA crosslinking and oxidative DNA base damage.
The technique is based on fluorescence microscopy and involves embedding nuclei
in agarose gel, then electrophoretic separation across an applied electric field.
The DNA is stained with a fluorescent dye, and subsequently imaged under an epifluorescence
microscope. The resulting structure is reminiscent of a comet, consisting of the
nucleus head, and tail of damaged DNA. During electrophoresis, fractured DNA fragments
migrate away from the nucleus and the extent of DNA damage can be quantified by
measuring the relative % DNA between head and tail. Importantly, the exact class
of DNA damage can be examined by careful control over cell preparation conditions.
For example, the extent of alkali treatment (pH control) that is used to convert
a DNA damage site into a DNA fragment (essential for Comet detection) can discriminate
between different types of strand breaks. Sites of DNA base modifications can be
converted to DNA fragments by introduction of enzymes that act specifically on such
lesions. Furthermore, DNA crosslinking and binding can also be detected by the Comet
assay, by determining the extent to which these interactions retard migration of
DNA. Since its "impact", Comet has become a core technique within fields or study
such as toxicology, apoptosis, DNA repair, aging, cell cycle analysis and free radical
biology.
Comet Detection
Comet slides are examined under a fluorescent microscope, often by widefield epifluorescence
microscopy. Widefield epifluorescence microscopy has developed into a universally
accessible technique for study of fluorescently labelled cells and tissues. Over
the last few decades, this drive has been accelerated by improvements in fluorescent
probes, labelling chemistry, optical instrumentation (such as filters and objectives)
and detector technology.
The widefield technique involves flood-illumination of the field of view by a wavelength
or small wavelength range (often though use of an excitation filter and arc lamp).
The stoke-shifted fluorescent emission transmits though the dichroic, that was initially
used to reflect the shorter wavelength excitation light onto the sample, gets optically
filtered once again by an emission filter (often called barrier filter), and focused
onto the camera detector.
EMCCD for Enhanced Comet
EMCCD is a novel detector technology for incorporation into your Comet set-up. EMCCD
cameras use an on-chip amplification technology that can be accessed to amplify
the signal above the read noise floor.
In light-starved instances, one simply needs to apply some EM gain to amplify the
previously undetectable signal into a respectable marker. The real beauty of the
EMCCD is in its flexibility - operate as a standard high Quantum Efficiency (QE)
CCD, or as a single photon sensitive power-horse when the signal is weak and/or
exposure times need to be reduced.
- EMCCD ultrasensivity facilitates markedly reduced dye photo-bleaching, maximizing
the amount of time a stained Comet specimen can be observed without loss in signal
strength or data confidence.
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