Mutagenesis with Ethidium bromide/NTG or EMS

BACKGROUND

Mutagenesis is a valuable technique in modern biology and genetics. It is the deliberate creation of genetic mutations in organisms. It enables scientists to investigate the effects of genetic changes on an organism’s traits, functions, and diseases. Mutagenesis, the deliberate introduction of mutations into the DNA sequence, provides vital insights into gene function, regulatory systems, and the underlying causes of genetic illnesses.

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Mutagenesis can be accomplished by a variety of means, including chemical, physical, and genetic methods. Chemical mutagens, such as ethyl methane sulfonate (EMS) or ethidium bromide (EtBr), alter DNA to cause mutations. Physical mutagens, such as X-rays and gamma rays, cause DNA damage and subsequent mutations. Genetic mutagenesis is the process of carefully introducing specific mutations using genetic engineering technologies like as CRISPR-Cas9.

Mutagenesis has a wide range of uses. It aids in the understanding of gene function and its role in complex biological processes in basic research. Mutagenesis is also important in crop improvement initiatives because it allows for the production of plants with desirable features such as disease resistance or higher yield. Mutagenesis also helps to understand genetic diseases by identifying genes involved in specific disorders and providing model systems for researching their underlying mechanisms.

While mutagenesis has enormous potential, it is critical to examine the hazards and ethical consequences. To protect researchers, the environment, and the species subjected to mutagenesis, proper safety procedures and adherence to ethical principles are critical.

Overall, mutagenesis is a versatile and vital tool that has transformed our understanding of genetics and continues to propel advances in biology and medicine.

Mutagenesis with EtBr and NTG

REQUIREMENTS

• Bacterial strains (S. halstedii and S. violaceusniger)
• Agarose gel
• TBE buffer
• Electrophoresis unit
• Ethidium bromide (EtBr)
• Distilled water
• Ultraviolet light source
• Tween 80
• Glass wool
• Whatman filter paper grade 1
• Glycerol
• Test tube
• Hickey-Tresner (HT) broth/agar
  • 20 g agar
  • 1 g yeast extract
  • 1 g beef extract
  • 2 g NZamine A
  • 10 g dextrin
  • 20 mg CoCl2*6H2O
  • 1000mL distilled water

PROCEDURE

1. Select bacterial strains S. halstedii and S. violaceusniger ^[1]
2. Proceed with these 2 strains for plasmid DNA extraction
3. For plasmid DNA extraction, 1% agarose gel in 0.5X TBE was electrophoresed at 180 V for 18 hours with a 30-60 sec switching time at 12˚ C using a contour-clamped homogenous electric field.
4. Gels were stained for 20 minutes at room temperature with 0.5 mg/ml ethidium bromide, destained by rinsing with distilled water, then examined under ultraviolet light.
5. Prepare spore suspension of S. halstedii and S. violaceusniger, and grow both strains on Hickey-Tresner (HT) agar at 28C.
6. The spores were harvested by scraping, suspended in 0.1% Tween 80, then vortexed for 5-10 min at room temperature.
7. The suspension was filtered through glass wool and Whatman filter paper grade 1, and the spore pellet was suspended in 20% glycerol.
8. The plasmid was attempted to be cured by inoculating the spore suspension into HT broth containing 10mM EtBr or 3.0 mg/l NTG. ^[1]
9. Incubate the culture at 28˚ C 200rpm for 1-2 weeks, after growth, serial dilution was performed and plated onto HT agar medium, incubate at 28˚ C for 48 hr.
10. Select presumptive aerial mycelium-negative (amy-)colonies as mutated. ^[4]

Mutagenesis with EMS

REQUIREMENTS

• Seeds (e.g., barley)
• Polyethylene mesh bag
• Beaker
• Distilled water
• EMS
• Dimethyl sulfoxide (DMSO)
• Blotting paper

PROCEDURE

1. Mix the required volumes of water and 2% (V/V) DMSO and autoclave at 120˚ C for 15 min at 15 psi, allowing to cool at room temperature. ^[2]
2. In laminar airflow, add 1 ml of EMS to the water-DMSO solution by using the 0.2 µm filter.
3. Take a healthy seed, make 5 batches, and leave 1 batch of seed as a control.
4. Add EMS solution to remaining seeds in 0.005 to 0.2 M solution concentration, and keep it for 2-3 hours at room temperature
5. Place the seed batches in a separate polyethylene mesh bag and labeled them as per the EMS concentration used and seal them properly.
6. Soak the seeds by placing the bags in the beaker containing distilled water and leave for 24 hr at room temperature.
7. Remove the bags and shake off excess water
8. Place bags in the beaker containing the EMS solution as per their mentioned concentration for 2-3 hours at room temperature ^[2]
9. Place bags in the distilled water for 2-3 hr to remove excess EMS
10. Place seeds on the blotting paper to remove excess water and dry them
11. Sow the seeds immediately after treatment in pots, and observe the growth.

CONCLUSION

The procedure involves bacterial strain selection, plasmid DNA extraction, and mutagenesis selection by using the EtBr and NTG mutagens, the mutation confirmed by the amy negative colonies selection.

The healthy seeds are treated with the EMS solution and sowed in soil and observe the plant growth with the control seeds

REFERENCES

1. Ismail Saadoun, Ahmed Elbetieha and Willard T Blevins, “Mutagenesis by ethidium bromide and N-methyl-N’-nitro-N-nitrosoguanidine in off-flavour compound producing strains of streptomyces”, Journal of Bioscience, December 1998.
2. Chikelu Mba, Rownak Afza, Souleymane Bado and Shri Mohan Jain, “Induced Mutagenesis in plants using physical and chemical agents”, Plant cell culture: Essential methods, 2010.
3. Jinjing Guo, Cheng Liu, Caifang Zhang, Jing Wang, Sa Wang, Xiaoxi Liu, Xiaojuan Gao and Xiuli Wu, “Nitrous Acid and Diethyl Sulfate, as Chemical Mutagenic Agents, to Improve the Biomass, Metabolites, Enzyme Activity, and Antioxidant Activity of Wild Phellinus igniarius”, Food Science and Technology Research, 2019.
4. Victoria L. Singer, Timothy E. Lawlor, Stephen Yue, “Comparison of SYBR green I nuclic acid gel stain mutagenicity and rthidium bromide mutagenicity in the Salmonella/ mammalian reverse mutation assay (Ames test)”, Mutation Research, 1999.

Also read:

• Survival curve with chemical mutagens
• Preparation of Boric Acid
• Test for carbohydrates
• Limit test for sulphate
• Synthesis of 1,3-substituted pyrazole from diarylhydrazone and vicinal diol
• Test for purity of water
• Swelling power of bentonite
• Limit test for iron
• Limit test for lead

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