BIOLOGY – CLASS XII

BIOTECHNOLOGY
Learning Goals
 Principles of Biotechnology
 Tools of Recombinant DNA Technology
 Processes of Recombinant DNA Technology

Biotechnology: The techniques of using live organisms or enzymes from organisms to

produce useful products and processes come under biotechnology.

The European Federation of Biotechnology (EFB) has given following definition of

biotechnology:

“The integration of natural science and organisms, cells, parts thereof, and molecular
analogues for products and services is called biotechnology.”

PRINCIPLES OF BIOTECHNOLOGY
The two core techniques that enabled birth of modern biotechnology are:

a. Genetic engineering: It involves the techniques to alter the chemistry of genetic

material, techniques to introduce the altered genetic material into host organisms;
in order to change the phenotype of the host organism.
b. Maintenance of sterile (microbial contamination-free) ambience during the process
so that only the desired microbe/eukaryotic cell is produced in large quantities.

Three basic steps in genetically modifying an organism are as follows:

a. Identification of DNA with desirable genes;

b. Introduction of the identified DNA into the host;
c. Maintenance of introduced DNA in the host and transfer of the DNA to its progeny.

TOOLS OF RECOMBINANT DNA TECHNOLOGY

Following are the key tools of recombinant DNA technology:
Restriction enzymes, polymerase enzymes, ligases, vectors, and the host organism

Restriction Enzymes
The enzymes which cut the DNA are called restriction enzymes. Hind II was the first
restriction endonuclease to be discovered. Hind II always cuts DNA molecules at a particular
point by recognizing a specific sequence of six base pairs. This specific base sequence is
called the recognition sequence for Hind II. Today, more than 900 restriction enzymes are
known. Different enzymes recognize different sequences.
Exonuclease: These enzymes remove nucleotides from the ends of the DNA.

Endonuclease: These enzymes make cuts at specific positions within the DNA.

Nomenclature of Restriction Enzymes: Each enzyme is named after the bacterium from
which it was isolated. The naming system is based on bacterial genus, species and strain.
Following table shows the name of EcoRI restriction enzyme and the implied naming
system.

Derivation of EcoRI name

Abbreviation Meaning Description

E Escherichia Genus

co coli species

R RY 13 Strain

I First identified Order of identification

Palindromes: A group of letters that form the same word when read both forward and
backward is called a palindrome. Restriction enzyme cut at the palindrome sequence in a
DNA. Following is an example of palindrome sequence:

5' —— GAATTC —— 3'

3' —— CTTAAG —— 5'

Restriction enzymes cut the strand of DNA a little away from the centre of the palindrome
sites, but between the same two bases on the opposite strands. This leaves single stranded
portions at the ends. There are overhanging stretches called sticky ends on each strand.
These are named so because they form hydrogen bonds with their complementary cut
counterparts. This stickiness of the ends facilitates the action of the enzyme DNA ligase.

When cut by the same restriction enzyme, the resultant DNA fragments have the same kind
of ‘sticky-ends’ and, these can be joined together (end-to-end) using DNA ligases.

Unless, the vector and the source DNA are cut with the same restriction enzyme, the
recombinant vector molecule cannot be created.

Separation and isolation of DNA fragments: The cutting of DNA by restriction

endonucleases results in the fragmentes of DNA. These fragments can be separated by a
technique known as gel electrophoresis. Since DNA fragments are negatively charged
molecules they can be separated by forcing them to move towards the anode under an
electric field through a medium/matrix.
Agarose is the most commonly used matrix. It is a natural polymer which is extracted from
sea weeds.

The DNA fragments separate according to their size through sieving effect provided by
agarose gel. So, smaller fragments move farther than longer fragments.

The separated DNA fragments are stained with ethidium bromide. After that, exposure to
UV radiation makes them visible. The DNA fragments appear as bright orange bands in this
case.

The separated bands of DNA are cut out from agarose gel and extracted; constructing
recombinant DNA by joining them with cloning vectors.

Cloning Vectors
A small piece of DNA; taken from a virus, a plasmid or the cell of a higher organism, that
can be stably maintained in an organism, and into which a foreign DNA fragment can be
inserted for cloning purposes; is called a cloning vector.

The following are the features that are required to facilitate cloning into a vector.

i. Origin of replication (ori): The sequence from where replication starts in the DNA is called
the Origin of Replication (ori). When a piece of DNA is linked to this sequence, it can be
made to replicate within the host cell. If you want to recover many copies of the target DNA,
it should be cloned in a vector whose ‘ori’ supports high copy number.
ii. Selectable marker: These are the substances which help in identifying and eliminating non-
transformants and selectively permitting the growth of the transformants. Generally, the
genes which encode resistance to antibiotics (ampicillin, chloramphenicol, tetracycline,
kanamycin, etc.) are considered useful selectable markers for E. coli. The normal E. coli cells
do not carry resistance against any of these antibiotics.
iii. Cloning sites: All cloning vectors have recognition sites for the commonly used restriction
enzymes. But to link the alien DNA, the vector needs to have very few, preferably single,
recognition site. Ligation of alien DNA is carried out at a restriction site present in one of
the two antibiotic resistance genes.
Example: A foreign DNA can be ligated at the Bam H I site of tetracycline resistance gene
in the vector pBR322. The recombinant plasmids will lose tetracycline resistance due to
insertion of foreign DNA but they can still be selected out from non-recombinant ones by
plating the tranformants on ampicillin containing medium.

After that, the transformants (growing on ampicillin containing medium) are transferred on
a medium which contains tetracycline.

The recombinant will grow in ampicillin containing medium but not in tetracycline
containing medium.

But non-recombinants will grow in medium containing both the antibiotics. In this case,
one antibiotic resistance gene helps in selecting the transformants, while other antibiotic
resistance gene gets inactivated due to insertion of alien DNA. Thus, it helps in selection of
recombinants.

Note: Selection of recombinants due to inactivation of antibiotics requires simultaneous

plating on two plates having different antibiotics. Hence, it is a cumbersome process.

Alternate selectable markers have been developed which can differentiate recombinants
from non-recombinants on the basis of their ability to produce color in the presence of
chromogenic substrate.

In this case, a recombinant DNA is inserted within the coding sequence of an enzyme, α-
galactosidase. This results into inactivation of the enzyme. (It is called insertional
inactivation).

The presence of chromogenic substrate gives blue-colored colonies if plasmid in bacteria

does not have an insert.

Presence of insert results into insertional inactivation of α-galactosidase, and no color is

produced by the colonies. Such colonies are identified as recombinant colonies.

Vectors for cloning genes in plants and animals: Bacteria and viruses have been
transferring their genes to transform eukaryotic cells; in order to force them to do what the
bacteria or viruses want. Now, these tools of pathogens can be transformed into useful
vectors for delivering beneficial genes to humans. For example; the tumor inducing (Ti)
plasmid of Agrobacterium tumifaciens has now been modified into a cloning vector which
is no more pathogenic to the plants. This vector can now be utilized to transfer beneficial
genes into many plants.

Competent Host (For Transformation with Recombinant DNA)

We know that DNA is a hydrophilic molecule. Hence, it cannot pass through cell
membranes. In order to force bacteria to take up the plasmid, the bacterial cell needs to
be ‘competent’’ to take up DNA. This is done by treating the bacterial cells with a specific
concentration of a divalent cation, e.g. calcium. It increases the efficiency with which DNA
enters the bacterium through pores in its cell wall.

Recombinant DNA can then be forced into such cells in following steps:

 Cells along with recombinant DNA are incubated on ice.

 They are then briefly placed at 42°C (heat shock).
 Then they are put back on ice.

Micro-injection: In this method, recombinant DNA is directly injected into the nucleus of
an animal cell.

Gene Gun: This method is applied for plant cells. In this method, cells are bombarded with
high velocity micro-particles of gold or tungsten coated with DNA.

Disarmed Pathogen: When the disarmed pathogen infects the cell, it transfers the
recombinant DNA into host.
GENE GUN – BIOLISTICS