“During agarose gel electrophoresis, The EtBr is used to visualize the DNA.
Not only in gel electrophoresis but also the EtBr is used in the karyotyping process of cytogenetics. In the present article, we will discuss the role of EtBr in both techniques.
The EtBr- ethidium bromide is a nucleic acid tagging agent that also functions as an antitrypanosomal drug in cattle during the early 19th century.
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 was unclear in killing kDNA.
Chemical name: Ethidium bromide
Abbreviation: EtBr, etbr, EB
Chemical formula: C21H20N3Br
Molecular weight: 394.4
Color emission: orange
EtBr is an aromatic molecule and has a heterocyclic moiety. Due to the intercalating property of EtBr, it is applied as an intercalating agent in molecular biology. Though the EtBr is most popular in gel electrophoresis of DNA, it has notable applications in cytogenetics too.
We have covered an entire series on agarose gel electrophoresis. please read all the articles here,
Role of EtBr in gel electrophoresis:
Agarose gel electrophoresis is a technique that can separate biomolecules like DNA, RNA, or proteins based on their charges. To separate DNA, under the influence of current, the negatively charged DNA migrates towards the positive charge.
DNA fragments separate in gel matrices based on their size and charge. Because our DNA doesn’t have their own color, we are not able to visualize it by the naked eye.
The EtBr works as a color agent that gives color to DNA.
EtBr works as a separating agent in agarose gel electrophoresis. EtBr intercalates between DNA base pairs and emits fluorescence under UV light. By using a standard orange filter, the orange-colored DNA can be seen. It emits fluorescence at 470nm wavelength.
EtBr has a tricyclic phenanthridine ring system. This ring interacts with the hydrophobic interior of the DNA bases with a strong van der Waals bond. Roughly one EtBr molecule will binds per 2.5 base pairs of DNA.
The hydrophobic environment present in between the basepairs increases the intensity of the fluorescence. Water is a highly efficient quencher of fluorescent. After intercalation between base pairs, under the hydrophobic environment, the EtBr removes water as it moves away from the buffer and emits fluorescence, increasingly.
With increasing the concentration of EtBr, the intensity of fluorescence increases. The standard concentration of EtBr for gel electrophoresis is 0.5μg/ml for 50ml of gel.
As it stacks between the bases of double-stranded DNA, we can’t identify single-stranded DNA by EtBr. PCR products and other genomic DNAs are visualized and identified by using EtBr during electrophoresis.
Furthermore, restriction digestion products, RNA, and various DNA fragments can be identified using it.
Notably, EtBr can change DNA properties like mobility, molecular weight and charge.
Sigmon J and Larcom LL in 1966, had studied the effect of EtBr intercalation on double-stranded DNA. They had used various concentrations of EtBr to check the mobility of DNA.
As the concentration of EtBr increases the mobility of DNA decreases. You can read their research paper here: The effect of ethidium bromide on the mobility of DNA fragments in agarose gel electrophoresis.
Their finding suggests that intercalated EtBr alters the conformational and physical properties of DNA. In spite of that, it is frequently used for DNA confirmation studies. We can use EtBr in two ways:
- Direct addition of EtBr into the gel
- Gel staining by EtBr
The first method is widely accepted across all molecular laboratories.
A desired amount of agarose is dissolved in a buffer and boiled until a clear solution appears. Once the temperature becomes touchable, add EtBr in the solution and mix it well by gentle shaking.
Though the present method is widely applicable, it has several limitations.
The activity of EtBr decreases at a higher temperature therefore the fluorescence level decreases. Furthermore, EtBr can’t spread well if the solution becomes colder quickly.
In the second method, after the completion of a gel run, the gel is overnight stained with the mild EtBr solution. This method is time-consuming and not recommended because of the carcinogenic nature of EtBr.
Role of EtBr in cytogenetic:
Cytogenetics is a study of chromosome structure, function, and related abnormality. Here, using karyotyping the metaphase chromosomes are cultured and fixed to detect chromosomal abnormalities.
The conventional cytogenetics method is remarkably popular to detect various syndromes such as Turner syndrome, Down syndrome, Patau syndrome, and Klinefelter syndrome.
Usually, the metaphase chromosomes are more condensed thus minor various can not be interpreted well. The EtBr releases chromosomal condensation and makes is longer.
It appears longer than normal chromosomes which is suitable in screening minor variation.
In cytogenetics, the present method is known as high-resolution banding using which smaller deletions, duplications, and insertions can be distinguished.
We can increase the number of chromosomal bands by adding EtBr before harvesting chromosomes.
Chromosomes are cultured for 72 hours and harvested afterward. EtBr can be added at the 70th hour of harvesting.
As we explained, once it is added, it intercalates between DNA bases and prevents DNA folding and condensing. The Uncondensation of DNA results in makes protein unable to fold DNA properly.
The loosely packed DNA makes the chromosome appear longer and increases band sizes. As the numbers of chromosome bands increase the accuracy of the result increases.
However, some scientists believe that EtBr does not have any significant role in the elongation of chromosomes.
Shinichi Misawa et.al., in 1986 proposed a finding that EtBr does have an important role in chromosomal elongation. Cultured human bone marrow cells were treated with different doses of EtBr before two hours of harvesting.
When compared without the EtBr culture results, the yield of the result was 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 chromosomes in high-resolution banding.
We have covered an article on the condensation process of a chromosome and DNA packaging. Read the article here: DNA packaging in eukaryotes
Limitations of EtBr
- EtBr is a fluorescent dye and has less self-life.
- As it can bind with DNA, it is mutagenic.
- EtBr is carcinogenic and teratogenic in nature. 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 over a period of time.
My ultimate guide for the handling of EtBr
EtBr is carcinogenic, teratogenic and mutagenic in nature. We should follow some safety guidelines while handling EtBr.
- 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 during the addition of EtBr and result analysis.
- Remove electrophoresis tank buffer every time after completion of each run. Follow proper EtBr disposal guidelines.
- Discard gloves after handling EtBr.
- Do not dispose of EtBr with regular chemicals. It is highly mutagenic and carcinogenic therefore place it in a separate sealed container and follow waste disposal management guidelines for EtBr or do as indicated by manufacturers protocol.
Indeed, the role of EtBr in molecular genetics and cytogenetics is significant, I personally prefer to use EtBr during Karyotyping and I always get a good quality of karyograms. Next time when you will perform Karyotyping add EtBr at the 70th hour of culture and believe me you will get good results.
Sybr Green is the best alternative to EtBr and it is safe as compared with EtBr. However, Sybr Green cannot be useful in karyotyping. So if you are working on molecular genetics and cytogenetics, the EtBr will be the best cost-effective option for you.
Article written by: Dr Tushar Chauhan
Article reviewed by: Binal tailor