BIOTECHNOLOGY – PRINCIPLES AND PROCESSES

CHAPTER-2

BIOTECHNOLOGY – PRINCIPLES AND PROCESSES

It is the technique of using living organisms or enzymes from organisms to produce a
product or processes useful to humans. Biotechnology emerged as a result of marriage between
biological sciences with technology.

Genetic Engineering involves the alteration of genetic constitution of cells or individuals.

Genetic Engineering or recombinant DNA technology is essentially ‘cut’, ‘copy’ and ‘paste’
mechanism. The gene to be transferred is first cut out from the DNA of donor organism. It is
then pasted into the DNA of vector. The vector carries the gene into the host organism where it is
copied many times as the host cell replicate. The advantage of Genetic Engineering technology
from hybridization is only the desirable genes are introduced into the target organism.

The cutting of DNA is done with the help of restriction enzymes (molecular scissors).
The cut piece of DNA is then linked to the plasmid (extra chromosome) DNA. The plasmid act
as a vector to transfer the desired DNA into the host organism. The cut piece of DNA is joined
by an enzyme, DNA ligase (molecular glues). This plasmid with desirable foreign DNA is then
transferred into the host cell where it multiplies and produces numerous copies.

RAJESH BABU.B .S, PKSHSS KANJIRAMKULAM

 BIOTECHNOLOGY – PRINCIPLES AND PROCESSES

Tools of rDNA (recombinant DNA)Technology: -The different tools used in rDNA technology
are restriction enzymes, polymerase enzyme, ligases, vectors and host cell.

(i) Restriction enzymes: - It belongs to large classes of enzymes called nucleases. There are two
kinds of nucleases exonucleases and endonucleases. Exonucleases cut nucleotides at the end of
the DNA.

Endonuclease may produce random cuts within the DNA. But restriction endonuclease
produce cut at a particular site called restriction site. The restriction endonuclease recognizes the
palindromic DNA sequence.

(In palindrome the nucleotide sequence are complementary strands that read the same in opposite
direction.)

Eg:-

Restriction enzymes makes two types of cut in DNA molecule sticky end cut and blunt end cut.
In sticky end cut they cut the two strands of DNA at two points. So there is one end is
overhanging. Such overhanging complementary single strand regions are called sticky ends or
cohesive ends.

Some restriction enzymes cut DNA across a straight line. So the cut ends remains blunt.

RAJESH BABU.B .S, PKSHSS KANJIRAMKULAM

 BIOTECHNOLOGY – PRINCIPLES AND PROCESSES

The first restriction endonuclease enzyme is Hind-II.

(ii) Polymerase Enzymes:-DNA polymerase enzymes help in the replication of DNA. It helps to
synthesize complimentary DNA strand on DNA template.

(iii) Ligases: - The cut produced by the restriction enzymes are joined by the ligase enzymes. So
they are also called molecular glues.

(iv) Vectors: - These are the agents that carry the insect DNA into the target site. When
introduce into a living cell a vector can replicate and produce many identical copies of the
inserted gene. Eg:- PBR322,Cosmid (Artificial plasmid created from Phage)

A cloning vector must have some common features.

(i) It must have an origin of replication that is recognized by the host cell.
(ii) It must have a cloning site where a desired DNA can be inserted.
(iii) Presence of a selectable marker (marker gene)

(v)Host cell: - These are bacterial cells which accept the rDNA through vector. Inside the host
cell the rDNA multiplies to form a clone.

Process of Recombinant DNA Technology: -rDNA Technology involves several steps

(i) Isolation and purification of DNA

(ii) Fragmentation of DNA by restriction endonucleases.
(iii) Isolation of desired DNA fragment (Electrophoresis)
(iv) Amplification of desired DNA (PCR)
(v) Ligation of the desired DNA fragmentinto a vector
(vi) Transfer the recombinant DNA into the host
(vii) Culturing the host cell and isolation of the desired product.

1.Isolation and purification of DNA:-In order to cut the DNA with restriction enzymes it must
be in pure form. DNA is enclosed within the membranes. First we have to break the cell to
release DNA along with other macromolecules, such as RNA, proteins, polysaccharides and
lipids. For breaking the cell it must be treated with particular enzymes. For breaking the bacterial
cell lysozyme enzyme is used, for plant cell cellulase enzyme is used, for fungal cell chitinase
enzyme is used. The RNA can be removed by treating the extracted DNA with ribonuclease,
proteins can be removed by protease. Similarly, by adding proper enzymes all impurities are
removed from DNA. Finally pure DNA is precipitated out by the addition of chilled ethanol.

RAJESH BABU.B .S, PKSHSS KANJIRAMKULAM

 BIOTECHNOLOGY – PRINCIPLES AND PROCESSES

2.Fragmentation of DNA by restriction endonucleases: - The purified DNA is treated with

restriction enzymes. That will produce cut at specific locations. The fragmented DNA contains
both desired and undesired DNA fragments.

3.Isolation of desired DNA fragment (Electrophoresis):- The desired DNA fragment can be
separated by a technique called gel electrophoresis. DNA molecules are negatively charged. So
they can be separated by forcing them to move towards the anode under an electric field through
a medium or matrix. The most commonly used medium is Agarose (It is a natural polymer
extracted from sea weeds.) The DNA fragments for separation is loaded in the wells of agarose
gel(pits of the gel) The DNA fragments separate according to their size through sieving effect
provided by the agarose gel. So smaller fragments will separate away from the pit and larger
fragments near the pits. The separated DNA fragments can be visualized only after staining the
DNA with a compound known as ethidium bromide and followed by exposure to UV light. The
DNA is now seen as bright orange coloured bands. The separated bands of DNAare cut out from
the agarose gel and extracted from the gel piece. It is known as elution.

4.Amplification of desired DNA (PCR):- It is one of the cell free DNA cloning technique. In
this the multiple copies of the desired gene or DNA is synthesized using two sets of primers
(Small chemically synthesized oligonucleotides that are complementary to the region of DNA)
and DNA polymerase enzyme. There are three steps in Polymerase Chain Reaction (PCR).

(A) The first step in the PCR is the thermal denaturation of the DNA sample by raising the
temperature up to 95 o C. This temperature is maintained for one minute. In addition to the
temperature we give two sets of primers, 4 deoxyribo nucleotides and DNA polymerase. It will
break the hydrogen bonds of the double stranded DNA.

(B) In the second step the temperature is slowly cooled to about 55 o C. During this step the
primers, base pair with the complementary sequence in the sample DNA. This process is called
annealing.

(C) In the third step the temperature is raised to 75o C. This temperature is suitable for the action
of DNA polymerase. The most commonly used DNA polymerase is Taq polymerase isolated
from a thermophilic bacterium Thermus aquaticus.The primer is essential for the initiation of
DNA synthesis. Primer extends in 51 31direction and two new double stranded DNA is
produced. This amplified fragment can now use to ligate with a vector for further cloning.

5. Ligation of the desired DNA fragment into a vector:-In order to link the desired DNA,
the vector DNA must have a recognition site for the restriction enzymes. The ligation of desired
DNA is carried out at a restriction site present in one of the two antibiotic resistant genes.

Eg: -PBR322

RAJESH BABU.B .S, PKSHSS KANJIRAMKULAM

 BIOTECHNOLOGY – PRINCIPLES AND PROCESSES

It is an artificially constructed plasmid of E.coli. It contains two antibiotic resistant gene

sites as marker i.e., Tetracycline, and Ampicillin. We can ligate the desired DNA at the Bam HI
site of tetracycline resistance gene in the PBR322, by using the ligase enzyme. The recombinant
plasmids will loss the tetracycline resistance due to the insertion of desired DNA.

6.Transfer the recombinant DNA into the host: - DNAis hydrophilic molecule but cell
membrane is hydrophobic. So the DNA does not pass through the cell membrane. So for
receiving the recombinant plasmid by a bacterial cell, it must be making suitable to receive the
plasmid. So the cell is treated with suitable divalent cation such as Ca 2+. It will increase the
permeability of the cell wall. Recombinant DNA is then forced into such cells by incubating the
cell on ice, and then give a heat shock (42o C) and then putting them back on ice.This enables the
bacteria to receive the recombinant DNA.

In another method, suitable for animal cell is the recombinant DNA is directly injected
into the cell. This method is called micro-injection. In plant cell the recombinant DNA is
transferred by coating the r DNA with gold or tungsten and it is bombarded into the cell. It is
known as biolistic or gene gun.

Now we can test the transformed cell from non-transformed cell. Here is the importance
of two marker gene in the plasmid. When the desired gene that we ligate is enter in the
tetracycline site, it loss the tetracycline resistance. So the growth rate in tetracycline medium is
less. But the transformed (r plasmid) cells have fast growth in ampicillin medium. The non-
transformed cell shows fast growth in both the antibiotics tetracycline and ampicillin. So the
marker helps to differentiate the transformed cell from non-transformed.

An alternative selectable marker has been developed, which differentiate the

recombinants from non- recombinants. It is based on the ability to produce colours in the
presence of a chromogenic substrate. In this the desired DNA is inserted in the coding sequence
of an enzyme β- galactocidase. This will result in the inactivation of enzyme called insertional
inactivation. The presence of the chromogenic substrate gives blue coloured colonies if the
plasmid in the bacteria does not have the desired DNA. Presence of desired DNA in the plasmid
result insertional inactivation of β- galactosidase and the colonies not produce the colour. These
are identified as recombinant colonies.

Similar to the transfer of genes in prokaryotes now we can transfer a desired gene into a
eukaryotic plant cell and animal cell using suitable vectors. Agrobacterium tumifaciens is a
pathogen of several dicot plants. It transfers its DNA called T- DNA into the plant cell and
transforms the normal plant cell into tumor cell. This T- DNA directs the cells toproduce the
chemicals needed by the pathogen. This nature of Agrobacterium is useful to deliver a gene of
interest into the host cell. The Ti-plasmid of Agrobacterium tumifaciens is now modified into a
cloning vector which is no more pathogenic to plant but is used to deliver the gene of interest
into a variety of plants.

RAJESH BABU.B .S, PKSHSS KANJIRAMKULAM

 BIOTECHNOLOGY – PRINCIPLES AND PROCESSES

Similarly, Retro virus affect animal cells and transform it into cancerous.
Retro virus is also disarmed and is now used to deliver desirable genes into animal cells.

7. Culturing the host cell and isolation of the desired product: - The hybrid cell is now
cultured in a suitable medium to produce large biomass for producing the desired protein or
product. To produce large quantities of proteins, bioreactors are used. Bioreactors are large
vessels in which raw materials are biologically converted into products. The most commonly
used bio-reactor is stirring type. The bio-reactor has an agitator system, oxygen delivering
system, foam control system, temperature control system, and P H control system. The culture is
taken in the bio-reactor for getting the product. After getting the product from bio-reactor it
should be separated and purified for marketing. It is called Down Stream Process. The product is
then added with suitable preservatives. After strict quality control test for each product it is
marketed.

Additional Information

1. PBR322 Hybrid plasmid of E.coli. Selective markers Amp R and TetR

2. PACYC177 Hybrid plasmid of E.coli. Selective markers Amp R and KanR
3. PWWO Hybrid plasmid of Pseudomonas putida. Selective marker Kan R
4. Bam H1- Restriction enzyme acting site of Bacillus amyloliquefaciens
5. Sal- 1 Restriction enzyme site of Streptomyces albus.
6. Hind III Restriction enzyme acting site of Haemophilous influenza.
7. PAGE – Poly Acrylamide Gel Electrophoresis.
8. 51 end – Phosphate terminal
9. 31 end – Hydroxyl terminal

RAJESH BABU.B .S, PKSHSS KANJIRAMKULAM