[Biology Class Notes] on Insertional Inactivation Pdf for EXAM

A recombinant DNA technique called inactivation involves the insertional inactivation of DNA. By using this method, recombinant plasmids or fragments of foreign DNA are added to bacteria to insert into a restriction site inside a gene to be resistant to antibiotics. This results in the gene becoming inactive or non-functioning as a result.

What is Meant by Insertional Inactivation?

Upon the insertion of a foreign gene into a pUC19 plasmid vector, the lacZ gene which encodes beta-galactosidase can no longer be produced. A fundamental process of screening and selection of recombinant DNA occurs once the molecule is inserted into the host cell when it becomes necessary to identify those cells containing the molecule.

Selection or screening are terms used to describe this process. In virtue of this, certain traits or characteristics are either not expressed or not expressed. The process of inactivating elements is an effective method of screening. During this procedure, foreign DNA is introduced to disturb one of the genetic characteristics. It is often referred to as the Blue-White selection method because it is widely used in the selection of recombinant plasmids for inactivation procedures.

An insert is made in the vector containing the lacZ gene, which is a reporter gene. There are a few restriction enzyme recognition sites in the β-galactosidase enzyme encoded by the lacZ gene. As a result of this reaction, a synthetic substrate X-gal, also called BCIG (5-Bromo-4-chloro-indolyl-β-D-galactopyranoside) is converted into an insoluble product with a blue color.

In the case of introducing a foreign gene to lacZ, the gene will be deactivated. Due to the deactivation of lacZ, no blue color will develop as no β-galactosidase will be produced. Thus, a host cell that contains rDNA tends to produce white-colored colonies on an X-gal medium, while a host cell carrying non-recombinant DNA tends to produce blue-colored colonies. Therefore, the color of the colony is used to select the recombinants.

Requirements in r-DNA Technology

In order to alter the DNA of an organism, certain tools must be used. Following are some examples.

• Restriction Enzymes- A number of enzymes have the ability to either cut a particular DNA strand or to add chemical groups to a specific DNA. A restriction endonuclease enzyme is responsible for cutting DNA. Restrictions Endonucleases attack DNA at specific points and do not randomly cut it. Instead, they cut DNA at these points when they come across such points. 

Example- Using EcoRI, DNA is cut at site GAATTC, where it is a restriction endonuclease enzyme.

• Ligase Enzymes- An enzyme such as this helps join a foreign DNA segment to DNA where changes need to be made or are being performed.

• Vectors- Recombinant DNA is transferred into the host organism by these organisms. By multiplying, cloning vectors produce a greater amount of recombinant DNA.

Example: Bacteriophages are a good example of this.

The Use of an Insertional Inactivation Process

In this process, recombinant DNA-containing organisms are distinguished from non-recombinant ones. Markers are selected based on their ability to determine recombinant DNA content.

Insertional Inactivation Method

The plasmid is the main component for carrying out the process of Insertional Inactivation. A plasmid has various genes present in it on different sites. These genes offer features like antibiotic resistance to the organism that incorporates them. A plasmid named pBR322 is considered for carrying out the process or method of Insertional Inactivation.

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This plasmid has two sites that give resistance to the antibiotics ampicillin and tetracycline respectively. By the technique of genetic engineering, a foreign gene is inserted in the site BamHI (site for tetracycline resistance). Now the recombinant plasmid will lose the resistance towards tetracycline as some other gene is inserted at its place. To recognize the recombinant the plating of ampicillin and tetracycline is used. Recombinant bacteria will grow in the ampicillin but will start dying in the tetracycline as they have lost tetracycline resistance.