Comet Assay is a type of gel electrophoresis
that is use to check the broken backbone or
broken segments of DNA using electrodes
and fluorescence stains to view the
damaged pieces. The whole procedure is
explained in the assignment.
Bacterial
Genetics
10-4-2023
Contents
Introduction: ......................................................................................................................................... 2
Protocol: ................................................................................................................................................ 2
Suspension: ........................................................................................................................................ 2
Lysis: .................................................................................................................................................. 2
Gel Electrophoresis: .......................................................................................................................... 2
Diagram: ................................................................................................................................................ 3
Comet Assay
Introduction:
Comet assay which is also known as the single-cell electrophoresis is a technique discovered in
late 1900’s. This technique is most frequently used now a days for the identification of broken
DNA fragments of eukaryotic cells. The ground work of this technique is the addition of cells with
agrose gel (as it is electrophoresis process). The mixture is than lysed using detergents and high
salts. This process allows the DNA to become a new form Nucleoids containing super coiled DNA
structures. The high pH level allows the broken DNA fragments to move towards the opposite side
(towards anode). The increased part over the cell identifies the amount or intensity of the broken
part of DNA known as comet.
Protocol:
The whole procedure of comet assay can be divided into 3 main parts.
Suspension
Lysis
Electrophoresis
Suspension:
For the formation of suspension, the cell is mixed with the low-melting agrose gel. It is very
important to take low melting point agrose because the low melting agrose is liquid at 37* C while
all other different types of agrose are solid at 37* C. The liquid agrose will allow the cell to mix
with agrose and form a suspension. The mixture is then poured on slides that are made specifically
for the comet assay, these are also known as the comet slides. These slides usually have a dig in
them to hold the material and are readily available. The comet slides after the pouring of
suspension are then left to solidify and then is sent for the Lysis procedure.
Lysis:
The prepared slides are then immersed in the Lysis solution. This solution contains high pH and
salt quantity and detergents. This solution allows the degradation of cell membrane and allows the
separation of DNA from histone protein. There will be a denaturation of the DNA that will help
the DNA strands to unwind and it will not cause any breakage. The Lysis solution will separate
out all the membranes, protein, cytoplasmic and nucleoplasmic content from the DNA. All the
material that is left behind DNA etc. are altogether known as the Nucleoids.
Gel Electrophoresis:
The electrophoresis is done as general electrophoresis but with electrodes attached to both end and
is attached to electric device. When the electrophoresis is run, there are certain bands that will have
a head and a tail. The head is a nucleus head and the tail tells about the breakage of DNA segment,
this head and tail altogether will form a comet. The tail will only form if the DNA strands contain
a breakage or damage in it. The damaged part will move towards the negative electrode forming
the tail like structure.
The structures are then stained with some fluorescence dyes and stains, so when these structures
are placed under the fluorescence microscope or a microscope that is connected with CCD and a
computer system in which these structures can be analyzed and the structures can be seen very
clearly as bright shining heads and tails.
Diagram:
