Biotechnology deals with the application of biology and biological concepts to science and engineering for the welfare of mankind. It is the crossroad of the biological sciences with other major disciplines of science, from organic chemistry to mechanical engineering. The history of biotechnology is as old as the history of man. When the first human beings realized that they could plant their own crops and breed their own animals, they learned to use biotechnology. But the term biotechnology was introduced by the end of the 20th century. Now a days the field of biotechnology becomes a vast field, but this chapter touches a brief part of biotechnology, called genetic engineering, which deals with manipulation or alteration in the genetic material of an organism.

CLONING OF GENES

Gene cloning is the act of making copies, or clones, of a single gene.

Once a gene is identified and cloned, it can be used in many areas of biomedical and industrial research.

 There are two possible ways of cloning of gene: recombinant DNA technology and polymerase chain reaction (PCR).

Recombinant DNA Technology

Recombinant DNA technology is a series of procedures that are used to join DNA segments from different sources.

This is an in vivo (in living cells) method which is used when gene cloning is required at industrial scale.

The main advantage of this method is the production of product of gene beside copies of gene.

It involves the selection and isolation of desired gene (gene of interest), inserting it in a suitable vector and the transformation of a suitable host by the recombinant DNA.

Components/Tools of Recombinant DNA Technology

The cloning of gene through recombinant DNA technology requires gene of interest, molecular scissors, molecular carrier or vector, molecular glue and expression system.

Gene of Interest

The gene of interest is the gene which is to be cloned. It can be obtained by one of the three possible ways:

(a) artificial gene synthesis is the process of synthesizing a gene in vitro (In glassware) without template DNA samples with the help of DNA synthesizer machine.

(b) Gene of interest can also be obtained by synthesizing it from its mRNA. Synthesis of gene ham mRNA is carried out by reverse transcriptase enzymes which are naturally found in retroviruses. The DNA formed by this process is called complementary DNA (cDNA).

(c) In most of the cases the gene of interest is directly cleaved from a chromosomal DNA by using particular DNA scissors called restriction endonucleases.

MOLECULAR SCISSORS/RESTRICTION ENDONUCLEASES

Restriction endonucleases are enzymes that cleave the phosphodiester bonds of both strands of duplex DNA at specific sequences.

In 1970, the first restriction enzyme was isolated. Many different restriction endonucleases have been isolated so far.

Naturally restriction enzymes are found in bacteria, where they appear to serve as host-defense role because they chop up and inactivate (“restrict”) the DNA of infecting viruses.

Each ‘restriction enzyme cleaves DNA at specific sequence of DNA called recognition sites or restriction sites.

These sites have palindromic sequences. A palindromic sequence is a four to eight base pairs in DNA in which nucleotides are arranged symmetrically in reverse order.

Restriction enzymes either make staggered cut or blunt cut. A staggered cut is one in which the resulting duplex fragments show single stranded projected ends called sticky ends.

While in blunt cut the resulting duplex, fragments do not show such sticky ends. In the fig 26.1, the recognition site is boxed in yellow and the cut sites indicated by red triangles.

MOLECULAR CARRIERS OR VECTORS

Vectors are another major component required to make a recombinant DNA (rDNA) molecule for gene cloning.

Vectors act as a vehicle for carrying foreign DNA into a host cell for multiplication.

Usually small circular DNA molecules of bacterial origin are used as cloning vectors.

A DNA molecule should possess the following essential characteristics to act as a cloning vector:

(a) Origin of replication site,

(b) antibiotics resistant genes,

(c) restriction sites of different enzymes.

Example of vectors are: Plasmid, lambda phage DNA, Cosmid (combination of plasmid and phage DNA), Yeast artificial chromosomes (YACs) etc.

MOLECULAR GLUE (DNA LIGASE)

This enzyme is responsible for the formation of the phosphodiester linkage between two adjacent nucleotides and thus joins two double-stranded DNA fragments; therefore, it is called molecular glue.

In rDNA experiments, DNA ligase is used to join two different DNA fragments (plasmid/vector and the foreign DNA) that are annealed by the sticky ends.

EXPRESSION SYSTEM

A suitable organism that can act as host for the recombinant vector to express (multiplication) is called expression system.

Therefore, the selection of suitable expression system always depends upon the type of vector which is being used while making recombinant DNA.

The most important character of an ideal expression system is its short generation time and simplicity of its genetic system. So bacterial cells can act as an ideal expression system.

MECHANISM OR PROCEDURE OF RECOMBINANT DNA TECHNOLOGY

Cloning of the desired gene through recombinant DNA technology involves the formation of recombinant DNA (gene of interest + vector DNA), transformation of a suitable expression system by the recombinant DNA, and the identification of transformed clones.

FORMATION OF RECOMBINANT DNA

The first step in the construction of a recombinant DNA, is the isolation and purification of vectors and gene of interest.

First, digest the vector DNA (e.g., plasmid) with same restriction enzyme by which gene of interest is cleaved so that compatible sticky ends can be produced.

Next both, vector and gene of interest are incubated together in the presence of DNA ligase which connects them by forming phosphodiester linkage.

This results in the formation of recombinant DNA molecule of vector and the gene of interest.

TRANSFORMATION OF EXPRESSION SYSTEM

Here transformation refers to the insertion of recombinant DNA into the expression system which can be performed by putting the expression system (bacterial cells that already contain no plasmids) and recombinant plasmids into the same medium.

Bacterial cells take up recombinant plasmid, especially, if they are treated with calcium chloride which make them more permeable.

Thereafter, as the cell reproduces, a bacterial clone forms and each new cell contains at least one plasmid.

Therefore, each of the bacterial cell contains the gene of interest, which will express itself and make its product.

IDENTIFICATION OF TRANSFORMED CLONE

The transformed clone can be identified by adding a particular antibiotic (for which resistant gene is found in plasmid) into the medium.

As the transformed clone has got resistance against the antibiotic, so it remains alive and continues to grow, whereas all the untransformed clones are killed by the antibiotic.

From this transformed bacterial clone, the cloned gene can be isolated for further analysis or its protein product can be separated and used for various purposes.