Monday, January 26, 2009

Understanding of Mutagenic Techniques

Mutations are heritable changes that occur in the genome or DNA. Normally, these changes are harmful to the organism, a small percentage of mutations are useful in the process of evolution. Mutations can be induced with the help of different mutagenic agents and chemical or physical methods to create improved qualities in microbial systems and in plants for producing improved crop varieties. For example, improved agronomic characteristics such as disease resistance, salt tolerance, early flowering, pest resistance, early maturing, grain size, etc.

Bacterial Mutagenesis: The best-suited method for inducing mutation in microbes such as bacteria is radiation, particularly UV-radiation.

The following is an experiment to induce mutation in bacteria with UV-radiation:

Step –1: Make a bacterial culture by inoculating a bacterial colony (s strain of e.coli) to a small volume of broth culture (liquid medium; LB medium or the minimal medium) and grow the culture overnight in an incubator shaker at 37°C.

Step –2: The next day, spread 0.1 ml of overnight culture on LB agar plates using a bent 'L' shaped glass rod (spreader) that has been flamed after being dipped in alcohol. Each plate should be labeled properly to avoid confusion before starting the experiment.

Step –3: Now keep the agar plate containing the bacteria to be mutagenized under the UV lamp in a laminar flow chamber or hood. Remove the cover from the plate and close the door of the hood. Turn the UV lamp on and note the time of exposure of the bacteria to the UV light. (Exposure should be timed in seconds. You can find out the optimal time of exposure by repeating the experiment between 5 and 240 seconds to determine what is optimal.)

The UV lamp is very strong. Do not expose your skin to the UV light. Do not operate the UV lamp with the hood open. UV light can cause serious and permanent damage to your eyes. The glass in the hood door will absorb the UV light. Never look at the UV lamp when it is on without wearing eye protection. At the end of the time turn the lamp off.

Step –4: Replace the cover on the plate, remove it from the UV box, and place it in a 37°C incubator. Plates should be incubated upside down. This is important to prevent accumulation of condensation of moisture on the surface of the agar.

Step –5: 'Check plates after 24 hours and count or estimate the number of colonies on the plate, and check for the type of mutant that you are looking for.

Depending on the type of mutant that you are looking for, prepare another set of the agar media plates to grow and select the mutants. If your aim is to get an auxotrophic mutant for Arginine (Arg-), you prepare agar media plates with minimal media, which contains only the essential components in the form of salts and elements and no organic components except the carbon source in the form of glucose. Now transfer the colonies from the master plates to the selection plate by replica plating. Take a circular filter paper that fits inside the petriplate and gently keep it over the colonies on the master plate. Slowly take the filter paper and put it in the selection media and allow growing overnight inside the incubator. By comparing the colonies in the selection media with that of the master plate, you can find out the colonies on the master plate, which cannot grow on the minimal media. This can be confirmed by monitoring the growth of the colonies again on the minimal media, which is supplemented with arginine (arg + plates).

Seed Mutagenesis: In the case of agricultural plants seeds are the parts that can be used for inducing mutations. We can use both chemical as well as physical mutagens for creating mutations.

For this experiment, we can use a mutagenic chemical-EMS (ethyl methanesulphonate) for inducing mutations in wheat or any other experimental plants such as arabidopsis. Take a specific number of healthy seeds and soak them in water overnight. The next day, the water is blotted off with tissue paper or filter paper. Incubate the seeds in an aqueous solution of EMS of suitable concentration for about 2 hours at room temperature. Incubate some seeds in water under similar physical conditions and use them as the control for the experiment. Note the concentration and the time of treatment of the experiment. After the exposure, take the seeds out and wash them thoroughly with water to remove the traces of the mutagen. The treated seeds have to be sown in the controlled environment along with the control separately. After germination, the seedlings of the treated seeds can be compared with that of the control to evaluate the desired mutations.

The experiment can be repeated with different strengths (concentration) of the mutagens and time of exposure.

Tags: Bio Technology, Bio Genetics, Mutagenic Techniques

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