Role of Ethidium bromide in agarose gel electrophoresis and Cytogenetics
Ethidium bromide is commonly used as a nucleic acid tagging agent in life science research. However, during the mid 19th century, EtBr was used as an anti-trapynosomal drug in cattle.
It was believed that it can kill the mitochondrial DNA, named kinetoplast (kDNA) and prevent cattle from the Trypanosoma infection. Still, the role of EtBr is unclear in killing kDNA.
Chemical name: Ethidium bromide
Abbreviation: EtBr, etbr, EB
Chemical formula: C21H20N3Br
Molecular weight: 394.4
Colour emission: orange
EtBr is an aromatic molecule and has a heterocyclic moiety. Due to the intercalating property of EtBr, it is used as an intercalating agent in molecular biology. Majorly, the EtBr is most popular in gel electrophoresis of DNA. However, it has routinely used in cytogenetics too.
In this article, We will discuss the role of EtBr in both techniques.
Role of EtBr in gel electrophoresis
Agarose gel electrophoresis is routinely used in the separation of molecules like DNA, RNA and protein. Under the influence of constant current, the negatively charged DNA migrates toward the positive charge.
The fragments of DNA is separated into gel metrics based on the size of DNA fragments. nonetheless, DNA does not have its own colour, hence it is impossible to visualize DNA without any colour.
EtBr work as a separating agent in agarose gel electrophoresis. EtBr intercalates between DNA base pairs and emits fluorescence under UV light. With the use of a standard orange filter, it emits orange colour at 470nm wavelength.
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EtBr contains the tricyclic phenanthridine ring system. This ring interacts with the hydrophobic interior of the DNA bases with the strong van Der walls bond. Approximately one EtBr molecule will binds per 2.5 base pairs of DNA.
Th hydrophobic environment present in between the basepairs increases the intensity of the fluorescence. Water is a highly efficient quencher of fluorescent. After intercalating between base pairs, under the hydrophobic environment, the EtBr removes water as it moves away from the buffer and emits fluorescence, increasingly.
Additionally, as the concentration of EtBr increases the intensity of fluorescence will increase. The standard concentration of EtBr for gel electrophoresis is 0.5μg/ml for 50ml of gel.
EtBr is used for the identification of PCR products specifically the double-stranded DNA. Single-stranded DNA can also visualise by EtBr.
Likewise, It is used for the identification of genomic DNA, restriction digestion products and RNA identification.
Intercalated EtBr changes several properties of DNA such as mobility, molecular weight and even the charge of DNA.
Sigmon J and Larcom LL in 1966, studied the effect of EtBr intercalating on double-stranded DNA. They used the different concentration of EtBr for the same fragment of DNA and each time as the concentration of EtBr increases the mobility of DNA fragment decreases.
Their finding suggests that intercalated EtBr alters the conformational and physical properties of DNA. In spite of that, it is mostly used in the confirmation studies (whether the fragment of our interest is presented or not).
Two different methods are used for addition of EtBr:
- Direct addition of EtBr into the gel
- Staining of the gel by EtBr
The first method is widely accepted across all molecular laboratories.
The required amount of agarose is dissolved into the buffer and boiled until a clear solution is obtained (all agarose is dissolved). After the temperature become touchable, EtBr can be added to the gel.
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In spite of the wide acceptance of this method, it has several common problems.
The activity of EtBr decreases at high temperature. Hence the fluorescence level can decrease. Additionally, if the agarose solution becomes colder quickly EtBr cannot be spread evenly into the gel.
In the second method, after the completion of a gel run, the gel is overnight stained with the mild solution of EtBr. This method is time-consuming and not recommended because of the carcinogenic nature of EtBr.
Role of EtBr in cytogenetic
“A study of chromosome structure, function and location” is called cytogenetics. The metaphase chromosome is visualized for the identification of structural or numerical anomalies in chromosomes.
EtBr plays a crucial role in the identification of chromosomes. The normal metaphase chromosomal plates are used for identification of numerical chromosomal abnormalities. Yet, we cannot identify smaller deletion or translocation.
Generally, metaphase chromosomes are more condensed as compare to other stages of cell division. Addition of EtBr releases the condensation of the chromosome and chromosome appears longer than the normal size.
Therefore it is easy to identify smaller deletions or addition. This technique is called as high-resolution banding. The number of the bands increases by the addition of EtBr before harvesting the chromosomes.
Generally, the chromosomes are cultured for 72 hours and then it is harvested. A couple of hours before harvesting (around ~70th hour), the EtBr can be added.
The loosely packed DNA makes the chromosome appear longer and the size of the bands can increase. As the numbers of bands increase the accuracy of the result will increases.
However, some scientist believes that EtBr does not have any significant role in the elongation of the chromosome.
Shinichi Misawa et.al., in 1986 proposed a finding that EtBr does have an important role in chromosomal elongation. Cultured human bone marrow cells are treated with different doses of EtBr before two hours of harvesting.
When compared without the EtBr culture, the yield of mitotic cells is increased by 2.9 folds and more than 400 bands per haploid cells were achieved by the addition of 10 μg/ml of EtBr.
EtBr treatment resulted in increasing the length of the chromosome by decreasing the condensation of the chromosome. Further, it increases the quality of chromosome in high-resolution banding.
Limitations of EtBr
- EtBr is fluorescent dye and has less shelf life.
- As it can bind with DNA, it is mutagenic.
- EtBr is carcinogenic and teratogenic. However, the carcinogenic property of EtBr is not proven. It affects through inhalation, skin absorption and ingestion.
- It can not be used for longer times as its activity decreases after several periods of time.
Handling and precautions
- Always prefer to use liquid and ready to use EtBr solution.
- While using EtBr, always wear gloves, glasses and a lab coat. Do not touch any of your body parts while handing EtBr.
- Always wear goggles while adding EtBr and during analysis of gel under UV light.
- Remove electrophoresis tank buffer each time after completion of each run. Follow proper EtBr disposal guidelines.
- Discard gloves after handling EtBr.
- Do not dispose EtBr with regular chemicals. It is a highly mutagenic and carcinogenic hence place it in a separate sealed container and follow waste disposal management guideline for EtBr or do as indicated by manufactures protocol.
Article written by: Tushar Chauhan
Article reviewed by: Binal tailor