**3.1 Gene therapy**

*Genetic Engineering - A Glimpse of Techniques and Applications*

Restriction endonucleases Cuts the DNA at specific nucleotide sequence DNA polymerase Addition of nucleotides at the 3′ end of the DNA

Exonucleases Remove nucleotide from the 3′ terminal of the DNA strand Alkaline phosphatase Removes phosphate group from both 5′ and 3′ terminals

DNA ligase Seals/joins the two fragments of DNA

**Enzymes Specific function**

*Enzymes involved in genetic engineering and their function.*

The mechanism of inserting the foreign DNA into host cell using a phage is termed as transduction. The virus can attach themselves on the host and transfer their DNA into the cells. In genetic engineering, this ability of viruses can be utilized purposefully for the transfer of recombinant DNA into the host cells. The recombinant DNA can be transferred to a virus by inserting it within any bacteria and infecting it with the bacteriophage [18]. The virus when multiplying within the cell also intakes the recombinant DNA, and after the completion of lytic cycle, the virus containing recombinant DNA bursts out the cell. This virus now contains recombinant DNA which can be transferred to the host cell by infecting it with the

Polynucleotide kinase Adds phosphate group to the 5′-OH end of the DNA to promote ligation

The mechanism by which the bacterial cell intakes DNA from the surrounding environment in which the bacteria is found or within the experimental solution is termed as transformation. This mechanism is carried out by exposing the bacteria to calcium chloride followed by heating the medium. This process will enable the bacteria to take up the recombinant DNA that we have introduced within their

It is a physical method in which electric pulse is applied across the cell which creates temporary pores in the cell membrane. The DNA is then transferred using these pores within the cells. In electroporation the host cells are introduced into a conductive medium, and an electric pulse at specific voltage is passed through the solution lasting for a few microseconds. The electric current alters the phospholipid layer of the membrane, and pores are generated through which foreign DNA is

It is a common method that is usually employed in transferring the recombinant DNA within any plant cell. In this process the DNA is injected physically in the cells using a gene gun. The microscopic particles of tungsten or gold are coated with the recombinant DNA and are loaded to a gun. The gun comprises of highpressure helium which ejects out the particles at a very high velocity. The outer

*2.3.1 Transduction*

**Table 1.**

phages.

*2.3.2 Transformation*

surroundings [15].

*2.3.3 Electroporation*

transferred [14].

*2.3.4 Microinjection*

**6**

Gene therapy is the process of correcting defective genes or introducing new genes into the existing cells for the cure and treatment of diseases [20]. Through gene therapy we can correct the root cause of the disease, i.e., the genes. The first approved gene therapy was employed to correct the deficiency of the enzyme adenosine deaminase (ADA) which was carried out on a 4-year-old girl Ashanti DeSilva. This girl suffered from severe combined immunodeficiency (SCID) as the gene coding for ADA was defective causing deoxyadenosine to accumulate and destroy T lymphocytes. After gene therapy, the 4-year-old developed no noteworthy side effects and grew normally into adulthood.

The gene therapy is of two types:


#### **3.2 Synthesis of insulin using genetically engineered** *E. coli*

In humans, insulin is produced as prohormone and needs to be processed to work as a functional hormone. Before the advent of genetic engineering, the insulin was extracted from the slaughtered cattle. This insulin when injected to the human patients in majority of cases induced allergies. The active human insulin comprises chains A and B linked together by disulfide bridges. The synthesis of insulin using genetic engineering was achieved by Eli Lilly, an American company in 1983 [5]. They isolated the human gene responsible for synthesis of insulin and introduced it into *E. coli* through a vector. The chains from the host cell were extracted, separated, and joined through disulfide bridges.

## **3.3 In agriculture**

The genetic engineering has revolutionized the agriculture sector and resolved the problem of feeding the ever-increasing population to a certain bit. Using genetic engineering different varieties of plants have been developed that possess better agronomic characteristics.

The crops developed through genetic engineering are termed as transgenic or genetically modified crops. The transgenic crops can be produced by transferring the gene of interest within the plant to obtain the desirable traits. The characteristics of plant can also be altered by silencing or removing their own genes.

The important characteristics of transgenic crops include:

