Comet Assay

Definition - What does Comet Assay mean?

Comet assay is a technique used to detect sperm DNA damage in a single sperm that may be contributing to male factor infertility. The comet assay is a highly sensitive method to identify breaks in the DNA of an individual sperm. It is helpful in predicting successful pregnancy. If more sperm display damaged DNA with the Comet assay, the chances of achieving a successful pregnancy, either spontaneous or through in vitro fertilization (IVF), are poor.

A Comet assay is also known as single-cell gel electrophoresis.

FertilitySmarts explains Comet Assay

Detection of sperm DNA damage is a useful tool for the evaluation of male factor infertility. The sperm contains a central nucleus that carries out all the genetic and growth activities of the cell. If this nucleus lacks protection against highly damaging molecules called free radicals, the sperm DNA is vulnerable to oxidative damage, which can be measured via the Comet assay.

The steps of the Comet assay are as follows:

1. Encapsulation

A sample of sperm cells is suspended in a low-melting-point agarose at room temperature. This suspension is then spread on a microscope slide. The agarose is next gelled at 4°C.

The agarose encapsulates the cells by developing a pattern of carbohydrate fibers. This helps secure the cells in place.

2. Burst

The slides containing the gelled agarose with sperm cell suspension are then immersed in a solution that renders the cells breakable and susceptible to destruction. The agarose matrix absorbs all the damaged components of the cell while leaving the DNA intact.

3. Electrophoresis

After the breakage of the cells, the slides are washed in distilled water and suspended in an electrophoresis solution. This solution serves to apply electric current to the cells. Electrophoresis at high pH yields structures that look like comets, as seen by fluorescence microscopy. The intensity of the comet tail gives an indication of the extent of DNA breaks.

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