*2.1.3 cDNA library*

*Genetic Engineering - A Glimpse of Techniques and Applications*

living cell thus altering the life process of the organism [9].

identified based on the altered properties of hybrid DNA [10].

**2.1 Isolation of desired DNA fragment or gene of interest**

*2.1.1 Production of DNA fragments by restriction digestion*

date, i.e., exonucleases and endonuclease [11].

purity using agarose gel electrophoresis.

*2.1.2 Genomic library*

which can be obtained by the methods or sources mentioned as follows:

process and its application in various fields.

**2. Steps involved in genetic engineering**

standing genetics.

transformed bacteria.

referred to as "protein engineering" since the biochemical properties of a protein

The methodology of manipulation of genetic material was developed in the 1970s. In vitro, the DNA was altered in a test tube and later introduced within the

The current chapter debriefs about the outline of the genetic engineering

In a broad perspective, manipulating the DNA is done by isolating it from the cells and cleaving it using sequence-specific restriction endonuclease. Further, the two independently isolated DNA from the microbial cells are mixed and sealed using DNA ligase. Lastly, the DNA is introduced into the cells, which are grown and

For example, DNA contains a gene that is responsible for providing antibiotic resistance to the microbial cell "A," isolated and introduced into a vector (plasmid), and then transferred into bacteria "B" which gains antibiotic resistance and is a

The first important step in genetic engineering is to acquire the gene of interest

The desired DNA fragment carrying the gene of interest is cleaved from the whole DNA using restriction enzymes. These enzymes are the key and an important base of genetic engineering. There are two types of restriction enzymes known till

Exonucleases cleave the dsDNA from the terminals, whereas endonucleases cleave the dsDNA at specific nucleotide sequence present amid the center. Different varieties of endonucleases with different cleavage sites have been identified and used in the process of genetic engineering. Certain restriction enzymes like EcoRI produce single-stranded self-complementary fragment with sticky ends, whereas

Many a times there exists a certain probability that the cleavage site of restriction enzyme is available within our gene of interest, and thus the gene will not remain whole after the restriction digestion [12]. This problem can be overcome by employing hydrodynamic forces to breakdown the DNA. Sonication and homogenization are the common methods employed for the fragmentation of DNA. The DNA fragment acquired by this method is purely random, and also no sticky ends or cohesive ends are generated. Later the DNA fragments are checked for size and

It comprises an entire genome of an organism that has been developed using molecular cloning methodology. The DNA of the organism is stored in population

enzymes like Hpa I produce double-stranded noncohesive fragments [8].

The technique of genetic engineering has evolved through our context of under-

are changed through gene mutation or in vivo alteration of genes [8].

**2**

This library comprises mRNA purified from a cell, tissue, or entire organism which has been changed back to dsDNA using reverse transcriptase. The cDNA/ complimentary DNA fragments are inserted into the host cell. A cDNA library comprises fragments of complimentary DNA which constitute certain portion of genome of the organism.
