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

EnzymesLigases Vectors

HOST ORGANIS

M

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

I 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:

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

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'

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 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'

Sticky and Blunt ends

Producing Recombinant DNA

First, the same REN is used to cut both the FOREIGN and VECTOR DNA’s 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.