**2. Steps involved in 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 identified based on the altered properties of hybrid DNA [10].

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 transformed bacteria.

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

The first important step in genetic engineering is to acquire the gene of interest which can be obtained by the methods or sources mentioned as follows:

#### *2.1.1 Production of DNA fragments by restriction digestion*

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

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

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 purity using agarose gel electrophoresis.

#### *2.1.2 Genomic library*

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

**3**

tant sites:

*Genetic Engineering: Altering the Threads of Life DOI: http://dx.doi.org/10.5772/intechopen.92618*

challenging.

*2.1.3 cDNA library*

genome of the organism.

chromosome (YAC), etc.

*2.2.1 The vector system*

of genetic engineering:

replicate within it.

foreign gene can be inserted.

promised after the insertion of a foreign gene.

**2.2 Insertion of gene into a suitable vector**

of identical vectors. In prokaryotes the genes coding for proteins are continuous, while in eukaryotes, exons (the coding region) are interrupted with introns (noncoding region). Thus, developing genomic library for eukaryotic organism remains

For construction of genomic library, the DNA from the organism is isolated and digested using restriction enzyme to get fragments of DNA of specific sizes. These fragments are later inserted into the vector using DNA ligase, and then the vector is introduced into a host organism which can be *E. coli* or yeast. The *E. coli* is a preferred host for protein production due to its rapid growth and ability to express proteins at high levels. It is also utilized in storing DNA sequences from other organisms. The genetically engineered *E. coli* is used for different studies of medical and pharmaceutical importance. Similarly, the yeast with altered genome has enhanced ability for the production of alcohol in brewery industry. The DNA fragment is later

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

A vector is a DNA molecule that has the ability to replicate inside the host to which the desired gene has integrated for cloning. The vectors include plasmids, cosmids, bacteriophages, bacterial artificial chromosome (BAC), yeast artificial

They are important cloning vehicles, within which the fragment of gene of interest is inserted and transferred into a suitable host system [11]. Mostly circular DNA that has small size and is of bacterial origin is utilized as vector. The vector should possess certain characteristics to make it suitable to be used for the process

• The vector DNA should possess the ability to infect the host organism and

• It must possess cleavage site for specific restriction enzyme through which a

• The ability of the vector DNA to replicate within the host should not be com-

• **Ori site**—The process of replication initiates from this sequence. The presence of this site is necessary for independent replication of plasmid within the host cell. This sequence is also responsible for controlling the copy

In order to promote cloning within vector, it must possess certain impor-

retrieved from the host cell for the purpose of analysis or study.

*Genetic Engineering: Altering the Threads of Life DOI: http://dx.doi.org/10.5772/intechopen.92618*

of identical vectors. In prokaryotes the genes coding for proteins are continuous, while in eukaryotes, exons (the coding region) are interrupted with introns (noncoding region). Thus, developing genomic library for eukaryotic organism remains challenging.

For construction of genomic library, the DNA from the organism is isolated and digested using restriction enzyme to get fragments of DNA of specific sizes. These fragments are later inserted into the vector using DNA ligase, and then the vector is introduced into a host organism which can be *E. coli* or yeast. The *E. coli* is a preferred host for protein production due to its rapid growth and ability to express proteins at high levels. It is also utilized in storing DNA sequences from other organisms. The genetically engineered *E. coli* is used for different studies of medical and pharmaceutical importance. Similarly, the yeast with altered genome has enhanced ability for the production of alcohol in brewery industry. The DNA fragment is later retrieved from the host cell for the purpose of analysis or study.
