What is recombinant DNA technology?

Recombinant DNA technology is the joining together of DNAmolecules from two different species. The
recombined DNA molecule is inserted into a host organism to produce new genetic combinations that
are of value to science, medicine, agriculture, and industry. Since the focus of all genetics is the gene,
the fundamental goal of laboratory geneticists is to isolate, characterize, and manipulate genes.
Recombinant DNA technology is based primarily on two other technologies, cloning and DNA
sequencing. Cloning is undertaken in order to obtain the clone of one particular gene or DNA sequence
of interest. The next step after cloning is to find and isolate that clone among other members of the
library (a large collection of clones). Once a segment of DNA has been cloned, its nucleotidesequence
can be determined. Knowledge of the sequence of a DNA segment has many uses.

When was recombinant DNA technology invented?

The possibility for recombinant DNA technology emerged with the discovery of restriction enzymes in
1968 by Swiss microbiologist Werner Arber. The following year American microbiologist Hamilton O.
Smith purified so-called type II restriction enzymes, which were found to be essential to genetic
engineering for their ability to cleave at a specific site within the DNA (as opposed to type I restriction
enzymes, which cleave DNA at random sites). Drawing on Smith’s work, American molecular
biologist Daniel Nathans helped advance the technique of DNA recombination in 1970–71 and
demonstrated that type II enzymes could be useful in genetic studies. About the same time, American
biochemist Paul Bergdeveloped methods for splitting DNA molecules at selected sites and attaching
segments of the molecule to the DNA of a virus or plasmid, which could then enter bacterial or animal
cells. In 1973 American biochemists Stanley N. Cohen and Herbert W. Boyer became the first to insert
recombined genes into bacterial cells, which then reproduced.

How is recombinant DNA technology useful?

Through recombinant DNA techniques, bacteria have been created that are capable of synthesizing
human insulin, human growth hormone, alpha interferon, hepatitis B vaccine, and other medically
useful substances. Recombinant DNA technology also can be used for gene therapy, in which a normal
gene is introduced into an individual’s genome in order to repair a mutation that causes a genetic
disease. The ability to obtain specific DNA clones using recombinant DNA technology has also made it
possible to add the DNA of one organism to the genome of another. The added gene is called a
transgene, which can be passed to progeny as a new component of the genome. The resulting organism
carrying the transgene is called a transgenic organism or a genetically modified organism (GMO). In this
way a “designer organism” is made that contains some specific change required for an experiment in
basic genetics or for improvement of some commercial strain.
Biotechnology: Principles and Processes

Processes of Recombinant DNA Technology

Recombinant DNA (rDNA) technology refers to the process of joining DNA molecules
from two different sources and inserting them into a host organism, to generate
products for human use. Can you put the DNA molecules in the host organism first
and then cut and join them? No! This process involves multiple steps that have to
proceed in a specific sequence to generate the desired product. Let’s understand
each step in detail.

Biotechnology and its Applications in Medicine

Introduction to Transgenic Organisms

Transgenic Micro Organisms

1. Isolation of Genetic Material

We already know that the genetic material of all living organisms is ‘nucleic acid’. In
most organisms, it is DNA, whereas in some it is RNA. The first step in rDNA
technology is to isolate the desired DNA in its pure form i.e. free from other
macromolecules.

However, in a normal cell, the DNA not only exists within the cell membrane, but is
also present along with other macromolecules such as RNA, polysaccharides,
proteins, and lipids. So, how do we break open the cell and obtain DNA that is free
from other macromolecules? We can use the following enzymes for specific purposes:

• Lysozyme – to break bacterial cell wall.

• Cellulase – to break plant cell wall.

• Chitinase – to break fungal cell wall.

• Ribonuclease – removes RNA.

• Protease – removes proteins (such as histones that are associated with DNA).
Other macromolecules are removable with other enzymes or treatments. Ultimately,
the addition of ethanol causes the DNA to precipitate out as fine threads. This is then
spooled out to give purified DNA.

2. Restriction Enzyme Digestion

Restriction enzymes act as molecular scissors that cut DNA at specific locations. These
reactions are called ‘restriction enzyme digestions’. They involve the incubation of the
purified DNA with the selected restriction enzyme, at conditions optimal for that
specific enzyme.

The technique – ‘Agarose Gel Electrophoresis’ reveals the progress of the restriction
enzyme digestion. This technique involves running out the DNA on an agarose gel. On
the application of current, the negatively charged DNA travels to the positive
electrode and is separated out based on size. This allows us to separate and cut out
the digested DNA fragments. The vector DNA is also processed using the same
procedure.

3. Amplification Using PCR

Polymerase Chain Reaction or PCR is a method of making multiple copies of a DNA
sequence using the enzyme – DNA polymerase. It helps to amplify a single copy or a
few copies of DNA into thousands to millions of copies. PCR reactions are run on
‘thermal cyclers’ using the following components:

• Template – DNA to be amplified

 • Primers – small, chemically synthesized oligonucleotides that are complementary
to a region of the DNA.

• Enzyme – DNA polymerase

• Nucleotides – needed to extend the primers by the enzyme.

Thermal Cycler [Source: Wikimedia Commons]

The cut fragments of DNA can be amplified using PCR and then ligated with
the cut vector as explained below.

4. Ligation of DNA Molecules

The purified DNA and the vector of interest are cut with the same restriction enzyme.
This gives us the cut fragment of DNA and the cut vector, that is now open. The
process of joining these two pieces together using the enzyme ‘DNA ligase’ is
‘ligation’. The resulting DNA is ‘recombinant DNA‘.

Restriction enzyme digestion followed by ligation. [Source: Wikimedia Commons]

5. Insertion of Recombinant DNA Into Host

In this step, the recombinant DNA is introduced into a recipient host cell. This process
is ‘Transformation’. Bacterial cells do not accept foreign DNA easily. Therefore, they
are treated to make them ‘competent’ to accept new DNA. (The topic – Tools of
Biotechnology explains a few ways to make cells competent).

During transformation, if a recombinant DNA bearing a gene for ampicillin resistance

is transferred into recipient E. coli cells, then the E. coli cells also become ampicillin-
resistant. This aspect is useful in differentiating transformed cells from non-
transformed cells.

For example, if we spread the transformed cells on agar plates containing ampicillin,
only the transformed, ampicillin-resistant cells will grow while the untransformed
cells will die. Therefore, in this case, the ampicillin resistance gene acts as the
‘selectable marker’.

6. Obtaining Foreign Gene Product

The recombinant DNA multiplies in the host and is expressed as a protein, under
optimal conditions. This is now a recombinant protein. Small volumes of cell cultures
will not yield a large amount of recombinant protein. Therefore, large-scale
production is necessary to generate products that benefit humans. For this purpose,
vessels called bioreactors are used.

Bioreactors are large containers with a continuous culture system, where the fresh
medium is added from one side and used medium is taken out from another side.
Bioreactors can process about 100-1000 litres of cell cultures. A bioreactor provides
optimum conditions (temperature, oxygen, pH, vitamins etc.) to biologically convert
raw materials into specific proteins, enzymes etc.

‘Stirred-tank bioreactor’ is the most common type of bioreactor. It is usually

cylindrical and has the following parts:

• Agitator system – to stir the contents evenly.

• Oxygen delivery system – to introduce air into the system.

• Foam control system

• Temperature control system

• pH control system

• Sampling ports – to take out small amounts of culture.

Bioreactor [Source: Wikimedia Commons]

7. Downstream Processing
Before the protein is marketed as a final product, it is subjected to downstream
processing which includes:

• Separation and purification.

• Formulation with suitable preservatives.

• Clinical trials to test the efficacy and safety of the product.

• Quality control tests.

Tools of Recombinant DNA technology

Inserting the desired gene into the genome of the host is not as easy as it
sounds. It involves the selection of the desired gene for administration into the
host followed by a selection of the perfect vector with which the gene has to be
integrated and recombinant DNA formed. This recombinant DNA then has to
be introduced into the host. And at last, it has to be maintained in the host and
carried forward to the offspring. A recombinant DNA technology can be
complete and achieved with the help of some elemental tools. The different
tools used for the purpose are discussed below:

Restriction Enzymes:
The enzymes which include the restriction endonucleases – help to cut, the
polymerases- help to synthesize and the ligases- help to bind.
The restriction endonucleases used in recombinant DNA technology play a
major role in determining the location at which the desired gene is inserted into
the vector genome. They are two types, namely endonucleases and
exonucleases. The endonucleases cut within the DNA strand whereas the
exonucleases cut the nucleotides from the ends of the DNA strands. The
restriction endonucleases are sequence-specific which is usually palindrome
sequences and cut the DNA at specific points. They scrutinize the length of
DNA and make the cut at the specific site called the restriction site. This gives
rise to sticky ends in the sequence. The desired genes and the vectors are cut
by the same restriction enzymes to obtain the complementary sticky notes, thus
making the work of the ligases easy to bind the desired gene to the vector.
Vectors:
The vectors help in carrying and integrating the desired gene. These form a
very important part of the tools of recombinant DNA technology as they are the
ultimate vehicles that carry forward the desired gene into the host organism.
Plasmids and bacteriophages are the most common vectors in recombinant
DNA technology that are used as they have very high copy number.
Host organism:
Host organism is the organism into which the recombinant DNA is introduced.
The host is the ultimate tool of recombinant DNA technology which takes in the
vector engineered with the desired DNA with the help of the enzymes. There
are a number of ways in which this recombinant DNA’s are inserted into the
host, namely – microinjection, biolistics or gene gun, alternate cooling and
heating, use of calcium ions, etc.

Restriction Endonuclease:

These enzymes serve as important tools to cut DNA molecules at specific sites, which is the
basic need for rec DNA technology.

These are the enzymes that produce internal cuts (cleavage) in the strands of DNA, only
within or near some specific sites called recognition sites/recognition sequences/ restriction
sites 01 target sites. Such recognition sequences are specific for each restriction enzyme.
Restriction endonuclease enzymes are the first necessity for rec DNA technology.

The presence of restriction enzymes was first of all reported by W. Arber in the year 1962. He
found that when the DNA of a phage was introduced into a host bacterium, it was fragmented
into small pieces. This led him to postulate the presence of restriction enzymes. The first true
restriction endonuclease was isolated in 1970s from the bacterium E. coli by Meselson and
Yuan.

Another important breakthrough was the discovery of restriction enzyme Hind-II in 1970s by
Kelly, Smith and Nathans. They isolated it from -the bacterium Haemophilus influenza. In
the year 1978, the Nobel Prize for Physiology and Medicine was given to Smith, Arber and
Nathans for the discovery of endonucleases.

Types of Restriction Endonucleases:

There are 3 main categories of restriction endonuclease enzymes:

Type-I Restriction Endonucleases

Type-II Restriction Endonucleases

Type-III Restriction Endonucleases

Type-I Restriction Endonucleases:

These are the complex type of endonucleases which cleave only one strand of DNA. These
enzymes have the recognition sequences of about 15 bp length (Table 1).

They require Mg++ ions and ATP for their functioning. Such types of restriction
endonucleases cleave the DNA about 1000 bp away from the 5′ end of the sequence ‘TCA’
located within the recognition site. Important examples of Type-I restriction endonuclease
enzyme are EcoK, EcoB, etc.

Type-II Restriction Endonucleases:

These are most important endonucleases for gene cloning and hence for rec DNA technology.
These enzymes are most stable. They show cleavage only at specific sites and therefore they
produce the DNA fragments of a defined length. These enzymes show cleavage in both the
strands of DNA, immediately outs.de then- recognition sequences. They require Mg++ions for
their functioning.

Such enzymes are advantageous because they don’t require ATP for cleavage and they cause
cleavage in both strands of DNA. Only Type II Restriction Endonucleases are used tor gene
cloning due to their suitability.

The recognition sequences for Type-II Restriction Endonuclease enzymes are in the form of
palindromic sequences with rotational symmetry, i.e., the base sequence .n the first half of
one strand of DNA is the mirror image of the second half of other strand of that DNA double
helix (Fig. 2). Important examples of Type-II Restriction endonucleases include Hinfl, EcoRI,
PvuII, Alul, Haelll etc.
Type-III Restriction Endonucleases:

These are not used for gene cloning. They are the intermediate enzymes between Type-I and
Type-II restriction endonuclease. They require Mg++ ions and ATP for cleavage and they
cleave the DNA at well-defined sites in the immediate vicinity of recognition sequences, e.g.
Hinf III, etc.

Nature of cleavage by Restriction Endonucleases:

The nature of cleavage produced by a restriction endonuclease is of considerable importance.

They cut the DNA molecule in two ways:

i. Many restriction endonucleases cleave both strands of DNA simply at the same point within
the recognition sequence. As a result of this type of cleavage, the DNA fragments with blunt
ends are generated. PvuII, Haelll, Alul are the examples of restriction endonucleases
producing blunt ends. Blunt ends may also be referred to as flush ends.

ii. In the other style of cleavage by the restriction endonucleases, the two strands of DNA are
cut at two different points. Such cuts are termed as staggered cuts and this results into the
generation of protruding ends i.e., one strand of the double helix extends a few bases beyond
the other strand. Such ends are, called cohesive or sticky ends.

Such ends have the property to pair readily with each other when pairing conditions are
provided. Another feature of the restriction endonucleases producing such sticky ends is that
two or more of such enzymes with different recognition sequences may generate the same
sticky ends.