Biotechnology

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

produce products and processes useful to humans.

Further, many other processes/techniques are also included under biotechnology. For
example, in vitro fertilisation leading to a ‘test-tube’ baby, synthesising a gene and using it,
developing a DNA vaccine or correcting a defective gene, are all part of biotechnology.

The definition given by EFB (European Federation Of Biotechnology) is as follows: ‘The


integration of natural science and organisms, cells, parts thereof, and molecular analogues
for products and services’.

Among many, the two core techniques that enabled the birth of modern biotechnology are :
(i) Genetic engineering : Techniques to alter the chemistry of genetic material (DNA and
RNA),to introduce these into host organisms and thus change the phenotype of the host
organism.
(ii) Maintenance of sterile (microbial contamination-free) ambience: in chemical engineering
processes to enable growth of only the desired microbe/eukaryotic cell in large quantities for
the manufacture of biotechnological products like antibiotics, vaccines, enzymes, etc.

The techniques of genetic engineering which include creation of recombinant DNA, use of
gene cloning and gene transfer, overcome this limitation and allow us to isolate and
introduce only one or a set of desirable genes without introducing undesirable genes into the
target organism.

Do you know the likely fate of a piece of DNA, which is somehow transferred into an alien
organism?
Most likely, this piece of DNA would not be able to multiply itself in the progeny cells of the
organism. But, when it gets integrated into the genome of the recipient, it may multiply and
be inherited along with the host DNA. This is because the alien piece of DNA has become
part of a chromosome, which has the ability to replicate. In a chromosome there is a specific
DNA sequence called the origin of replication, which is responsible for initiating replication.
Therefore, for the multiplication of any alien piece of DNA in an organism it needs to be a
part of a chromosome(s) which has a specific sequence known as ‘origin of replication’.
Thus, an alien DNA is linked with the origin of replication, so that this alien piece of DNA can
replicate and multiply itself in the host organism. This can also be called cloning or making
multiple identical copies of any template DNA.

The construction of the first recombinant DNA emerged from the possibility of linking a gene
encoding antibiotic resistance with a native plasmid (autonomously replicating circular
extra-chromosomal DNA) of Salmonella typhimurium. Stanley Cohen and Herbert Boyer
accomplished this in 1972 by isolating the antibiotic resistance gene by cutting out a piece of
DNA from a plasmid which was responsible for conferring antibiotic resistance. The cutting
of DNA at specific locations became possible with the discovery of the so-called ‘molecular
scissors’– restriction enzymes. The cut piece of DNA was then linked with the plasmid DNA.
These plasmid DNA act as vectors to transfer the piece of DNA attached to it. You probably
know that mosquitoes act as an insect vector to transfer the malarial parasite into the human
body. In the same way, a plasmid can be used as a vector to deliver an alien piece of DNA
into the host organism. The linking of antibiotic resistance genes with the plasmid vector
became possible with the enzyme DNA ligase, which acts on cut DNA molecules and joins
their ends. This makes a new combination of circular autonomously replicating DNA created
in vitro and is known as recombinant DNA. When this DNA is transferred into Escherichia
coli, a bacterium closely related to Salmonella, it could replicate using the new host’s DNA
polymerase enzyme and make multiple copies. The ability to multiply copies of antibiotic
resistance genes in E. coli was called cloning of antibiotic resistance genes in E. coli.
There are three basic steps in genetically modifying an organism —
(i) identification of DNA with desirable genes
(ii) introduction of the identified DNA into the host
(iii) maintenance of introduced DNA in the host and transfer of the DNA to its progeny.

TOOLS OF RECOMBINANT DNA TECHNOLOGY

genetic engineering or recombinant DNA technology can be accomplished only if we have


the key tools, i.e., restriction enzymes, polymerase enzymes, ligases, vectors and the host
organism.

1. Restriction Enzymes:
In 1963, the two enzymes responsible for restricting the growth of bacteriophage in
Escherichia coli were isolated. One of these added methyl groups to DNA, while the other
cut DNA. The latter was called restriction endonuclease.

The first restriction endonuclease–Hind II, whose functioning depended on a specific DNA
nucleotide sequence was isolated and characterised five years later. It was found that Hind II
always cut DNA molecules at a particular point by recognising a specific sequence of six
base pairs. This specific base sequence is known as the recognition sequence for Hind II.
Besides Hind II, today we know more than 900 restriction enzymes that have been isolated
from over 230 strains of bacteria each of which recognise different recognition sequences.

Restriction enzymes belong to a larger class of enzymes called nucleases. These are of two
kinds; exonucleases and endonucleases. Exonucleases remove nucleotides from the ends
of the DNA whereas endonucleases make cuts at specific positions within the DNA. Each
restriction endonuclease functions by ‘inspecting’ the length of a DNA sequence. Once it
finds its specific recognition sequence, it will bind to the DNA and cut each of the two strands
of the double helix at specific points in their sugar -phosphate backbones. Each restriction
endonuclease recognises a specific palindromic nucleotide sequence in the DNA.

These are groups of letters that form the same words when read both forward and
backward, e.g., “MALAYALAM”. As against a word-palindrome where the same word is read
in both directions, the palindrome in DNA is a sequence of base pairs that reads the same
on the two strands when orientation of reading is kept the same. For example, the following
sequence reads the same on the two strands in 5' à 3' direction. This is also true if read in
the 3' à 5' direction.
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.
Restriction endonucleases are used in genetic engineering to form ‘recombinant’ molecules
of DNA, which are composed of DNA from different sources/genomes.

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.

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


endonucleases results in the fragments 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. Nowadays the most commonly used matrix is
agarose which is a natural polymer extracted from sea weeds. The DNA fragments separate
(resolve) according to their size through the sieving effect provided by the agarose gel.
Hence, the smaller the fragment size, the farther it moves.
The separated DNA fragments can be visualised only after staining the DNA with a
compound known as ethidium bromide followed by exposure to UV radiation (you cannot
see pure DNA fragments in the visible light and without staining). You can see bright orange
coloured bands of DNA in an ethidium bromide stained gel exposed to UV light. The
separated bands of DNA are cut out from the agarose gel and extracted from the gel piece.
This step is known as elution. The DNA fragments purified in this way are used in
constructing recombinant DNA by joining them with cloning vectors.

2. Cloning Vectors:

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