Question

If you had a genomic library for a particular organism, what would you need to do in order to find a particular gene within that library? Explain in detail, showing that you understand the steps involved in this process.

Answer 1

The genomic library of an organism consists of all their genomic DNA. In this the DNA is stored in vectors and each vector carry different DNA insert. The genome library is constructed as given below.

1. The total DNA is extracted from the organism of interest.

2. The DNA will be subjected to restriction digestion using a restriction enzyme. In this step the DNA will cut into different fragments having a specific length. Each fragment will have one or two genes.

3. These fragments will be then inserted into a the vector which was previously cut using the same restriction enzyme. The enzyme DNA ligase is used to seal the insert into the vector.

4. The vectors carrying the DNA inserts will be transformed into a host organisms creating a DNA library.

For finding a particular gene of interest from the genomic library of an organism, the screening of the library should be performed. The genomic library screening is performed using technique called colony hybridization. Colony hybridization is a method used for selecting bacterial colonies containing desired gene of interest.

The important steps in finding a particular gene from a genomic library is given below.

1. The transformed bacteria from the genomic library are grown in nutrient agar plate.

2. The bacterial colonies are replicated symmetrically on a nylon or nitrocellulose membrane.

3. The cells on the membrane are then lysed and the DNA are dentured to make them single stranded.

4. A radiolabeled probe, which is complementary to the desired gene will be added onto the membrane and allow it to get hybridized to its complementary DNA sequence.

5. Wash off the unhybridized probes and expose the membrane to an X-ray film (autoradiography)

6. The DNA cluster that shows the gene of interest are then matched to the corresponding bacterial colonies on the agar plate. Then the positive clone carrying the desired gene can be isolated from the plate for further experiments.