Anmol Sharma

• Biotechnology is the technique of using live organisms or Biotechnology deals with:

their enzymes for products & processes useful to humans. - Microbe-mediated processes (making curd, bread, wine etc).
• The European Federation of Biotechnology (EFB) - In vitro fertilization (test-tube baby programme).
defines Biotechnology as ‘the integration of natural - Synthesis and using of a gene.
science and organisms, cells, parts thereof, and molecular - Preparation of DNA vaccine.
analogues for products and services’. - Correcting a defective gene.

PRINCIPLES OF BIOTECHNOLOGY
Core techniques of modern biotechnology c) Maintenance of introduced DNA in the host and
transfer of the DNA to its progeny: A piece of alien
• Genetic engineering: The technique in which genetic
DNA has no the sequence called Origin of replication
material (DNA & RNA) is chemically altered and
(ori) needed for starting replication. So, it cannot multiply
introduced into host organisms to change the phenotype.
itself in the progeny cells of the organism. Hence alien
• Bioprocess engineering: Maintenance of sterile ambience
DNA is integrated into the recipient genome (it has ori).
in chemical engineering processes for growing desired
It multiplies & inherits along with host DNA.
microbe/eukaryotic cell for the manufacture of antibiotics,
vaccines, enzymes etc. • The process of joining and inserting a foreign piece of
DNA into a host organism to produce new genetic
Basic steps in genetically modifying an organism combinations is called recombinant DNA technology.
a) Identification of DNA with desirable genes: Traditional • First recombinant DNA (rDNA) was produced by
hybridisation leads to inclusion and multiplication of Stanley Cohen & Herbert Boyer (1972).
undesirable genes along with desired genes. In genetic • They isolated an antibiotic resistance gene (piece of
engineering, only desirable genes are introduced. DNA) from a plasmid of Salmonella typhimurium. It was
b) Introduction of the identified DNA into the host: A linked with a plasmid vector and transferred into E. coli.
vector DNA such as plasmid is used to deliver an alien As a result, the gene was expressed & multiplied in E. coli.
piece of DNA into the host organism.

TOOLS OF RECOMBINANT DNA TECHNOLOGY

1. Restriction Enzymes (‘molecular scissors’) - Restriction endonuclease recognizes a specific
palindromic nucleotide sequences in the DNA. It is a
- The enzymes that cut DNA at specific sites into fragments.
sequence of base pairs that read the same on the two
- They belong to a class of enzymes called nucleases.
strands in 5' → 3' direction and in 3' → 5' direction. E.g.
- In 1963, two enzymes responsible for restricting growth of
Palindromic nucleotide sequence for EcoRI is
bacteriophage in E. coli were isolated. One enzyme added
5' —— GAATTC —— 3'
methyl groups to DNA. The other (restriction
3' —— CTTAAG —— 5'
endonuclease) cut DNA.
- More than 900 restriction enzymes have been isolated
from over 230 strains of bacteria.
Naming of the restriction enzymes:
- First letter indicates genus. The second two letters indicate
species of prokaryotic cell from which they were isolated.
E.g. EcoRI comes from E. coli RY 13 (R = the strain.
Roman numbers = the order in which the enzymes were
isolated from that strain of bacteria).
Types of Restriction enzymes:
• Exonucleases: They remove nucleotides from the ends of
the DNA.
• Endonucleases:
- They cut at specific positions within the DNA. E.g. EcoRI. Steps in formation of recombinant DNA by EcoRI
- They bind to specific recognition sequence of the DNA and
- Restriction enzymes cut the strand a little away from the
cut the two strands at specific points.
centre of the palindrome sites, but between the same two
- The first restriction endonuclease is Hind II. It cuts DNA
bases on the opposite strands. This leaves single stranded
molecules by recognizing a specific sequence of 6 base
overhanging stretches at the ends. They are called sticky
pairs. This is called the recognition sequence for Hind II.
ends. They form H-bonds with their complementary cut

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counterparts. This stickiness facilitates action of the - Ligation of alien DNA is carried out at a restriction site
enzyme DNA ligase. present in one of the two antibiotic resistance genes.
- When cut by the same restriction enzyme, the resultant E.g. In vector pBR322, foreign DNA is ligated at Bam H I
DNA fragments have the same kind of sticky-ends and site of tetracycline resistance gene. As a result,
these are joined together by DNA ligases. recombinant plasmid is formed. If ligation does not
occur, it is called non-recombinant plasmid.

• Restriction sites: Hind III,

EcoR I, BamH I, Sal I, Pvu
II, Pst I, Cla I.
• ori
• Antibiotic resistance
genes: ampR and tetR.
• Rop: codes for the
proteins involved in the
replication of plasmid.

- When a foreign DNA is inserted within a gene of bacteria,

that gene is inactivated. It is called insertional
inactivation. Here, the recombinant plasmids lose
tetracycline resistance due to insertion of foreign DNA.
- When the plasmids are introduced into E. coli cells, 3 types
2. Cloning Vector of cells are obtained:
o Non-transformants: They have no plasmid. So they
- It is a DNA molecule that can carry a foreign DNA
are not resistant to either tetracycline or ampicillin.
segment and replicate inside the host cells.
o Transformants with non-recombinant plasmid:
E.g. Plasmids, bacteriophages etc.
They are resistant to both tetracycline & ampicillin.
- Plasmids are autonomously replicating circular extra-
o Transformants with recombinant plasmid: They are
chromosomal DNA of bacteria. Some plasmids have only
resistant only to ampicillin.
1-2 copies per cell. Others have 15-100 copies per cell. - Recombinant plasmids can be selected out from non-
- Bacteriophages (high number per cell) have very high recombinant ones by plating transformants on ampicillin
copy numbers of their genome within the bacterial cells. medium. Then the transformants are transferred on
- When the cloning vectors are multiplied in the host, the tetracycline medium.
linked piece of DNA is also multiplied to the numbers - The recombinants grow in ampicillin medium but not on
equal to the copy number of the vectors. tetracycline medium. But, non-recombinants grow on the
Features required for cloning into a vector medium containing both the antibiotics.
a. Origin of replication (ori) - Thus, one antibiotic resistance gene helps to select the
- This is a sequence where replication starts. transformants. The inactivated antibiotic resistance gene
- A piece of DNA linked to ori can replicate within the host helps to select recombinants.
cells. This also controls the copy number of linked DNA. - But this type of selection of recombinants is a difficult
So, for getting many copies of the target DNA, it should be procedure because it needs simultaneous plating on 2 plates
cloned in a vector whose origin support high copy number. having different antibiotics. So, alternative selectable
b. Selectable marker (marker gene) markers have developed based on their ability to produce
colour in presence of a chromogenic substrate.
- It is a gene that helps to select the transformants and
- In this, a recombinant DNA is inserted into the coding
eliminate the non-transformants.
- If a piece of DNA is introduced in a host bacterium, it is sequence (gene) of an enzyme, b-galactosidase. So, the
called transformation. Such bacterium is transformant. If gene is inactivated (insertional inactivation). Such colonies
transformation does not take place, it is non-transformant. do not produce any colour. These are identified as
- Selectable markers of E. coli include the genes encoding recombinant colonies.
- If the plasmid in bacteria have no an insert, it gives blue
resistance to antibiotics like ampicillin, chloramphenicol,
coloured colonies in presence of chromogenic substrate.
tetracycline, kanamycin etc. Normal E. coli cells have no
resistance against these antibiotics. d. Vectors for cloning genes in plants & animals
c. Cloning sites Genetic tools of some pathogens can be transformed into
- These are the recognition sites for restriction enzymes. useful vectors for delivering genes to plants & animals. E.g.
- To link the alien DNA, the vector needs a single or very • Agrobacterium tumefaciens (a pathogen of many dicot
few recognition sites. plants) can deliver a piece of DNA (T-DNA) to transform
- More than one recognition sites generate several normal plant cells into a tumor. These tumor cells produce
fragments. It complicates the gene cloning. the chemicals required by the pathogen.
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The tumor inducing (Ti) plasmid of A. tumefaciens is through pores in cell wall → Incubate the cells with
modified into a cloning vector which is not pathogenic but recombinant DNA on ice → Place them briefly at 420C
can use mechanisms to deliver genes of interest into plants. (heat shock) → Put them back on ice → Bacteria take up
• Retroviruses in animals can transform normal cells into recombinant DNA.
cancerous cells. So, they are used to deliver desirable Other methods to introduce alien DNA into host cells
genes into animal cells. • Micro-injection: In this, recombinant DNA is directly
3. Competent Host (For Transformation with injected into the nucleus of an animal cell.
Recombinant DNA) • Biolistics (gene gun): In this, cells are bombarded with
- Since DNA is a hydrophilic molecule, it cannot pass high velocity micro-particles of gold or tungsten coated
through cell membranes. So bacterial cells are made with DNA. This method is suitable for plants.
• ‘Disarmed pathogen’ vectors: They infect the cell and
‘competent’ to take up alien DNA or plasmid as follows:
- Treat bacterial cells with a specific concentration of a transfer the recombinant DNA into the host. E.g. A.
divalent cation (e.g. calcium) → DNA enters the bacterium tumefaciens.

PROCESSES OF RECOMBINANT DNA TECHNOLOGY

1. Isolation of the Genetic Material (DNA) - Primers are small chemically synthesized oligonucleotides
that are complementary to the regions of DNA.
- Treat the bacterial cells/plant or animal tissue with enzymes
like lysozyme (bacteria), cellulase (plants), chitinase
Steps of PCR:
(fungus) etc. The cell is broken releasing DNA & other • Denaturation: It is the heating of target DNA (gene of
macromolecules (RNA, proteins, polysaccharides & lipids). interest) at high temperature (940 C) to separate the strands.
- RNA is removed by treating with ribonuclease. Proteins Each strands act as template for DNA synthesis.
are removed by treatment with protease. Other molecules • Annealing: It is the joining of the two primers (at 520 C)
are removed by appropriate treatments. at the 3’ end of the DNA templates.
- When chilled ethanol is added, purified DNA precipitates • Extension: It is the addition of nucleotides to the primer
out as a collection of fine threads in the suspension. using a thermostable DNA polymerase called Taq
polymerase. It is isolated from a bacterium, Thermus
2. Cutting of DNA at Specific Locations aquaticus. It remains active in high temperature during the
- Purified DNA is incubated with the restriction enzyme. denaturation of double stranded DNA.
As a result, DNA digests. These DNA fragments are Through continuous replication, the DNA segment is
separated by a technique called gel electrophoresis. amplified up to 1 billion copies.
The amplified fragment can be used to ligate with a vector
for further cloning.

- Agarose gel electrophoresis is employed to check the

progression of a restriction enzyme digestion. DNA is
negatively charged. So it moves towards the anode. DNA
fragments are separated according to their size through
sieving effect of the agarose gel (a polymer extracted from
sea weeds). The smaller sized fragment moves farther.
- The process is repeated with the vector DNA also.
- DNA fragments can be seen as bright orange coloured
bands when they are stained with ethidium bromide and
exposed to UV radiation.
- DNA bands are cut out from agarose gel. It is called
elution. The cut-out gene of interest and cut vector are 4. Insertion of Recombinant DNA into Host Cell
mixed and ligase is added. It creates recombinant DNA. - Using any methods, the ligated DNA is introduced into
3. Amplification of Gene of Interest using PCR recipient (host) cell / organism. They take up DNA from
- Polymerase Chain Reaction (PCR) is the synthesis of its surrounding.
multiple copies of the gene of interest in vitro using 2 sets - If a recombinant DNA bearing ampicillin resistant gene
of primers & the enzyme DNA polymerase. is transferred into E. coli cells, the host cells become
ampicillin-resistant cells.

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- If the transformed cells are spread on agar plates

containing ampicillin, only transformants will grow.
Untransformed recipient cells will die.
5. Obtaining the Foreign Gene Product
- The aim of recombinant DNA technology is to produce a
desirable protein.
- If a protein encoding foreign gene is expressed in a
heterologous host, it is called a recombinant protein.
- The cells with foreign genes can be grown in laboratory. It is usually cylindrical or with a curved base to facilitate the
The cultures are used to extract the desired protein and mixing of the reactor contents. The stirrer facilitates even
purify it by using separation techniques. mixing and oxygen availability. Alternatively, air can be
- The cells can also be multiplied in a continuous culture bubbled through the reactor.
system. Here, the used medium is drained out from one The bioreactor has
side while fresh medium is added from the other. It • An agitator system
maintains the cells more physiologically active and so • An oxygen delivery system
produces a larger biomass. It yields more desired protein. • A foam control system
Bioreactors • A temperature control system
- These are the vessels in which raw materials are • pH control system
biologically converted to specific products, enzymes etc., • Sampling ports (for periodic withdrawal of the culture).
using microbial, plant, animal or human cells.
6. Downstream Processing
- Bioreactors are used to produce large quantities of
products. They can process 100-1000 litres of culture. - It is a series of processes such as separation and
- A bioreactor provides the optimal growth conditions (pH, purification of products after the biosynthetic stage.
temperature, substrate, salts, vitamins, oxygen) to get - The product is formulated with suitable preservatives.
desired product. Such formulation undergoes thorough clinical trials and
- The most commonly used bioreactors are of stirring type strict quality control testing.
(stirred-tank bioreactor).

MODEL QUESTIONS
1. Identify the tools.
a) Separation of DNA b) Amplification of DNA
c) Large scale purification of product d) Isolation of separated DNA fragments
2. Draw & label the parts of pBR322.
3. Some processes of recombinant DNA technology are given below. Arrange them in correct order.
a. Amplification of gene of interest using PCR b. Cutting of DNA at specific locations
c. Obtaining the foreign gene product d. Insertion of recombinant DNA into the host cell
e. Isolation of the genetic material (DNA) f. Downstream processing
4. Observe the following and answer to the questions.
5’ GAATTC 3’
3’ CTTAAG 5’
a) Identify the above sequence.
b) What is the significance of this kind of sequence in recombinant DNA technology?
5. Restriction enzymes & ligases opened the doorway for recombinant DNA technology. Do you agree with this? Justify.
6. Electrophoresis is the migration of charged particles in solution under the influence of an electric field.
a) Who developed this technique? b) Name the supporting media in AGE and PAGE.
7. A plasmid is a circular double-stranded extra chromosomal DNA in a bacterial cell.
a) Name the naturally occurring plasmids in E. coli and in Agrobacterium.
b) Name an artificially reconstructed plasmid.
8. PCR is meant for making multiple copies of a gene of interest. Mention the major steps involved in PCR. Name an
organism form which a thermostable DNA polymerase is isolated.