Biotechnology:

Principles and Processes

Definition

It deals with techniques of using live organisms or enzymes from organisms to

produce products and processes useful to humans

European Federation of Biotechnology: It is the integration of natural

science and organisms , cells, and parts thereof, and molecular analogues for
products and services.

Principles of Biotechnology

Genetic Engineering: Techniques to alter the chemistry of genetic material

(DNA and RNA). Creation of RECOMBINANT DNA, use of GENE CLONING AND
GENE TRANSFER come under genetic engineering.

Maintenance of sterile conditions in chemical engineering processes to enable

the growth of desired microbe or cell in large quantities.

The construction of the first recombinant DNA emerged from the possibility of
linking a gene encoding antibiotic resistance with a native PLASMID, an
autonomously replicating circular extra-chromosomal DNA of S.typhimutium.

Basic Steps in Genetically Modifying an

Organism

Identification of DNA with desirable genes

Introduction of the identified DNA into the host

Maintenance of introduced DNA in the host and transfer of the DNA to its
progeny.

Tools of Recombinant DNA Technology:

Key tools

Restriction
Enzymes

Polymerase
Enzymes

Ligases

Vectors

HOST
ORGANISM

Tools of Recombinant DNA Technology:

Restriction Enzymes

A restriction enzyme or restriction endonuclease is an enzyme that cuts

DNA at or near specific recognition nucleotide sequences.

There are four types of REN and are classified according to their mode of
activity:

Type

Mode of Activity

Cuts DNA at random away from

restriction sites

II

Cuts at defined sequence or near

recognition site

III

Cuts outside restriction sites

IV

Cuts modified DNA

Contd

Each REN recognizes specific PALINDROMIC NUCLEOTIDE SEQUENCES in the

DNA and cut the two strands of the double helix in their sugar-phosphate
backbones.

Contd

In 1963, two enzymes responsible for restricting the growth of the E.coli
bacteria were isolated one added methyl groups to DNA, the other cut DNA.
The latter was called RESTRICTION ENDONUCLEASE.

The first REN was Hind II, isolated from H.influenzae, whose functioning
depended on a specific DNA nucleotide sequence, called recognition
sequences or restriction sites. For Hind II, it is:
5' G T ( pyrimidine: T or C) ( purine: A or G) A C 3'
3' C A ( purine: A or G) ( pyrimidine: T or C) T G 5'

It was found that Hind II cut directly at the centre of this sequence.

Another REN that was isolated from the

E.coli bacteria was EcoRI. The nucleic acid
sequence where the enzyme cuts is GAATTC.

The enzyme cuts both DNA at the same time,

and between the bases G and A only when
the above sequence is present.

The DNA fragments join at the sticky ends

to give recombinant DNA.

Sticky and Blunt ends

Sticky ends are so named because they form hydrogen bonds with their
complementary counterparts. This stickiness of the ends facilitates the
action of the action of DNA ligase.

There is another type of REN called Smal isolated from Serratia marcescens.It
produces blunt ends.

Blunt ends are the simplest end of a double stranded molecule, where both
strands terminates in a base pair. For example:

5'-CTGATCTGACTGATGCGTATGCTAGT-3'
3'-GACTAGACTGACTACGCATACGATCA-5'

Producing Recombinant DNA

First, the same REN is used to cut

both the FOREIGN and VECTOR
DNAs at specific points.

Then, DNA ligases joins the

FOREIGN DNA or the DNA
SEQUNCE OF INTEREST to the
VECTOR.

This RECOMBINANT DNA is then

passed onto further generations.

Separation and Isolation of DNA

Fragments: Gel Electrophoresis

The cutting of DNA by restriction endonucleases results in fragments of DNA.

These fragments can be separated by a technique known as GEL

ELECTROPHORESIS.

In this process, an agarose gel, a polymer extracted from seaweeds, is used as

a medium or matrix.

An electric field is applied and the DNA fragments separate or resolve

according to their size through the sieving effect provided by the agarose.

The separated DNA fragments are

stained by a compound, known as
ETHIDIUM BROMIDE, followed by
exposure to UV radiation.

Bright orange colored bands of DNA are

seen under UV light.

These bands are then cut out and the

DNA strands extracted from the gel
and are joined with cloning vectors.
This step is called ELUTION.