Are you curious to know what is insertional inactivation? You have come to the right place as I am going to tell you everything about insertional inactivation in a very simple explanation. Without further discussion let’s begin to know what is insertional inactivation?
Insertional inactivation is a technique used in molecular biology to disable or “knock out” a specific gene in a microorganism. It involves the insertion of a foreign DNA fragment into the target gene, disrupting its function. In this blog post, we will explore the concept of insertional inactivation in detail.
What Is Insertional Inactivation?
Insertional inactivation is a molecular biology technique used to knock out a specific gene in a microorganism. It involves the insertion of a foreign DNA fragment into the target gene, disrupting its function. This can be achieved by using a vector, which is a DNA molecule that can carry foreign DNA fragments into the host cell. The vector is designed to carry a selectable marker gene, which enables the selection of cells that have taken up the foreign DNA fragment.
Stages Of Insertional Inactivation
The process of insertional inactivation involves the following stages:
- Designing the vector: The first stage of insertional inactivation involves designing the vector that will carry the foreign DNA fragment. The vector must contain a selectable marker gene that allows the selection of cells that have taken up the foreign DNA fragment.
- Inserting the foreign DNA fragment: The next stage involves inserting the foreign DNA fragment into the vector. This can be achieved by using restriction enzymes, which cut the DNA at specific sites, and ligases, which join the DNA fragments together.
- Introducing the vector into the host cell: The vector is then introduced into the host cell, which can be a bacterial cell or a eukaryotic cell. The host cell takes up the vector, and the foreign DNA fragment is integrated into the genome of the host cell.
- Selection of cells: The final stage involves selecting cells that have taken up the foreign DNA fragment. This is done by using a selective medium that allows the growth of cells that have the selectable marker gene but not those that do not have it. The cells that have taken up the foreign DNA fragment will be unable to produce the protein encoded by the target gene, and thus will be unable to grow on the selective medium.
Importance Of Insertional Inactivation
Insertional inactivation is an important tool in molecular biology research. It enables the study of the function of specific genes by disrupting their function. It can also be used to create recombinant DNA molecules that can be used for various purposes, such as the production of recombinant proteins and the development of gene therapies.
In conclusion, insertional inactivation is a molecular biology technique used to knock out a specific gene in a microorganism. It involves the insertion of a foreign DNA fragment into the target gene, disrupting its function. Insertional inactivation is an important tool in molecular biology research, as it enables the study of the function of specific genes and the creation of recombinant DNA molecules for various purposes.
What Is An Example Of Insertional Inactivation?
For example, the insertion of a piece of foreign DNA into a cloning site that is located on an antibiotic-resistant gene on the vector can lead to the loss of the antibiotic resistance phenotype by insertional inactivation.
What Is The Insertional Inactivation Of The Lac Z Gene?
In the field of recombinant DNA technology, inactivation by insertion is a common process. Nevertheless, the lacZ gene gets inactivated when it is inserted into a foreign gene, such as a recombinant plasmid. The deactivation of the lacZ gene results in no blue-color colonies developing.
How Is The Insertional Inactivation Of An Enzyme Used As A Selectable Marker?
The recombinant or transformed organisms are unable to produce any color when grown on the chromogenic substrate. Thus, the insertional inactivation of an enzyme acts as a selectable marker to differentiate recombinants from non – recombinants.
Why Is Insertional Inactivation Utilized With Pbr322?
The insertional inactivation of pBR322 will result in a loss of resistance to tetracycline by E. coli. This can be found out by growing the recombinants on two plates; one containing tetracycline and another containing ampicillin. The recombinant will grow in ampicillin but not in tetracycline.
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