Introduction to recombinant DNA technology

Recombinant DNA technology (genetic engineering) refers to the joining together of DNA molecules from
two different species that are inserted into a host organism to produce new genetic combinations that are of
value to science, medicine, agriculture, and industry. Recombinant DNA (rDNA), on the other hand, is the
general name for a piece of DNA that has been created by the combination of at least two strands. They are
DNA molecules formed by laboratory methods of genetic recombination (such as molecular cloning) to
bring together genetic material from multiple sources, creating sequences that would not otherwise be found
in the genome.
Laboratory geneticists' primary purpose is to identify, define, and modify genes. Consider that each human
cell has about 2 meters (6 feet) of DNA (the human nuclear genome has approximately 3 200 000 000
nucleotides). As a result, even a small piece of tissue can contain thousands of kilometers of DNA.
Recombinant DNA technology, on the other hand, has made it possible to isolate a single gene or other
section of DNA, allowing researchers to establish its nucleotide sequence, investigate its transcripts, alter
it in extremely specific ways, and reintroduce the transformed sequence into a living creature.
History of recombinant DNA technology
While the structure of DNA was first determined in 1953, it wasn’t until the early 1970 years that the
first recombinant DNA molecules were produced by means of restriction enzymes. Paul Berg (Stanford)
succeeded in proving the possibility to splice and to recombine genetic material in 1971. Recombinant
DNA in a living organism was first achieved in 1973 by Herbert Boyer, of the University of California at
San Francisco, and Stanley Cohen, at Stanford University, who used E. coli restriction enzymes to insert
foreign DNA into plasmids– a subsequently submitted patent for recombinant antibody technology was
approved in 1980.

(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. Thus, the recombinant DNA has to be
introduced into the host. And at last, it has to be maintained in the host and carried forward to the offspring)
Tools Of Recombinant DNA Technology
The enzymes which include the restriction enzymes help to cut, the polymerases- help to synthesize and
the ligases- help to bind. The restriction enzymes used in recombinant DNA technology play a major role
in determining the location at which the desired gene is inserted into the vector genome. There are two
types, namely Endonucleases and Exonucleases.

The Endonucleases cut within the DNA strand whereas the Exonucleases remove the nucleotides from
the ends of the strands. The restriction endonucleases are sequence-specific which are 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.

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 a very high copy number. The vectors are made up of an origin of
replication- This is a sequence of nucleotides from where the replication starts, a selectable marker –
constitute genes which show resistance to certain antibiotics like ampicillin; and cloning sites – the sites
recognized by the restriction enzymes where desired DNAs are inserted.

Host 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 these recombinant DNAs are inserted into the host, namely –
microinjection, biolistics or gene gun, alternate cooling and heating, use of calcium ions, etc.
Steps in recombinant DNA production
Recombinant DNA is composed of sequences that are derived from different sources. The process to
achieve this involves the following steps:

1. Identification and Isolation of genetic material

2. Cutting of DNA at specific locations (GOI, Vector, Host cell)
3. Joining of DNA fragments by ligation and homopolymer tailing
4. Amplification
5. Insertion of DNA into the host cell
6. Selection and screening of transformed cells
The last step can be achieved by an immunological method or nucleic acid hybridization, blue-white
screening or insertional inactivation.
1. DNA isolation
DNA of interest is identified first (depending what do you want do to with it) and isolated in its pure form,
which means they are devoid of other macromolecules.
In rDNA technology, the initial step is to extract the desired DNA in its purest form, that is, free of
extraneous macromolecules. Because DNA coexists with other macromolecules such as RNA,
polysaccharides, proteins, and lipids within the cell membrane, it must be separated and purified using
enzymes such as lysozymes, cellulase, chitinase, ribonuclease, and proteases. Other enzymes or treatments
can remove other macromolecules. The DNA eventually precipitates out as fine threads as a result of the
presence of ethanol. After that, the pure DNA is spooled out.
2. Cutting of DNA/Restriction Enzyme Digestion
For this step, the restriction enzymes are quite vital. It helps to identify the location wherein a designated
gene is introduced into a vector genome. The said reaction is known as restriction enzyme digestion.
They entail incubating pure DNA with a restriction enzyme of choice at conditions that are appropriate for
that enzyme.
The Agarose Gel Electrophoresis technology displays the restriction enzyme digestion's progress.
This method entails passing the DNA across an agarose gel. When current is applied, negatively charged
DNA flows to the positive electrode and is divided into different sizes. This permits the digested DNA
fragments to be separated and snipped out.
The same method is used to process the vector DNA.
3. Amplifying of DNA
Copies of genes are amplified through PCR or polymerase chain reaction. It is essentially a process to
increase a single DNA copy into several copies after the desired gene of interest is cut with restriction
enzymes.
It allows a single copy or a few copies of DNA to be amplified into thousands or millions of copies.
The following components are used in PCR reactions that are conducted on 'thermal cyclers':
• Template: DNA that has to be amplified.
• Primers: oligonucleotides are tiny, chemically produced oligonucleotides that are complementary
to a DNA region.
• Enzyme: DNA polymerase (such as Taq polymerase). The enzyme is required to lengthen the
primers.
• Nucleotides (dNTPs):
PCR can be used to amplify the cut DNA fragments, which can subsequently be ligated with the cut vector.
4. Joining DNA
The vector and a section of DNA are joined in this step. It is achieved with the help of the enzyme DNA
ligase. With the same restriction enzyme, the pure DNA and the vector of interest are cut. This yields the
cut DNA fragment and the cut vector, both of which are now open. Ligation is the process of putting these
two parts together with the enzyme DNA ligase. The resulting DNA molecule is a hybrid of the interest
molecule and the vector DNA molecules. Recombination is the term used in genetics to describe the
merging of different DNA strands. As a result, this new hybrid DNA molecule is known as a recombinant
DNA molecule (rDNA), and the process is known as recombinant DNA technology.
5. Insertion of rDNA into a Host
Here rDNA is added to the recipient host cell, and the entire process is called transformation. Post insertion,
the recombinant DNA multiplies and manifests as manufactured protein under favorable conditions.
The recombinant DNA is then transferred into a recipient host cell, most commonly a bacterial cell, in this
stage. The term for this procedure is 'Transformation.' Bacterial cells have a hard time accepting foreign
DNA. As a result, they are given treatments to make them 'capable' of accepting new DNA (competent cell
preparation). Thermal shock, Ca++ ion therapy, electroporation, and other procedures may be applied.
6. Recombinant Cell Isolation
A mixed population of converted and non-transformed host cells results from the transformation process.
Only the transformed host cells are filtered during the selection procedure. The marker gene of the plasmid
vector is used to distinguish recombinant cells from non-recombinant cells.
PBR322 plasmid vector, for example, comprises two marker genes (Ampicillin resistant gene and
Tetracycline resistant gene). When grown on media containing a particular antibiotic, the antibiotic
resistance gene from the plasmid is expressed, causing the recombinant cells to become
Ampicillin/tetracycline resistant and non-transformants are Ampicillin/tetracycline sensitive.

Examples of recombinant DNA technology in

Health sector
The first commercial healthcare product derived from rDNA was human insulin. Today, it is successfully
applied to make new antibodies, vaccines (e. g. for Hepatitis B), and different protein production systems,
for instance for insulin and human growth hormone.
Agriculture
Recombinant DNA technology is used to genetically modify plants in order to improve adaptability as well
as resistance to harmful agents and to enhance product yield.

• Bt Crops: Bt crops are genetically modified to express a bacterial gene from Bacillus thuringiensis
(Bt), which produces a protein toxic to certain insect pests. These crops, such as Bt cotton and Bt
corn, offer built-in insect resistance, reducing the need for chemical insecticides and improving crop
yield.
• Golden Rice: Golden Rice is a genetically engineered variety of rice that produces beta-carotene,
a precursor of Vitamin A. This biofortified rice aims to combat Vitamin A deficiency in populations
relying heavily on rice as a staple food.
• Roundup Ready Crops: Roundup Ready crops, like soybeans and corn, are designed to be resistant
to the herbicide glyphosate. This allows farmers to control weeds more effectively, as the crops can
withstand glyphosate treatment, reducing the environmental impact of herbicides.
Food industry
Recombinant DNA technology enables the manufacturing of novel enzymes that are suitable to prolong
shelf life and kill foodborne pathogens.
• Bioengineered Food Additives: Recombinant DNA technology is employed to produce various
food additives and enzymes. For instance, certain enzymes used in food processing, such as
amylases and proteases, can be manufactured using rDNA methods, ensuring consistent and safe
production.
 DNA/gene Cloning
A clone is a cluster of individual entities or cells that are descended from one progenitor. Clones are
genetically identical as the cell simply replicates producing identical daughter cells every time. Scientists
are able to generate multiple copies of a single fragment of DNA, a gene which can be used to create
identical copies constituting a DNA clone.
DNA/gene cloning is is a molecular biology technique of making multiple, identical copies of a particular
piece of DNA. In a typical DNA cloning procedure, the gene or other DNA fragment of interest (perhaps a
gene for a medically important human protein) is first inserted into a circular piece of DNA called
a plasmid. The insertion is done using enzymes that “cut and paste” DNA, and it produces a molecule
of recombinant DNA, or DNA assembled out of fragments from multiple sources.
Next, the recombinant plasmid is introduced into bacteria. Bacteria carrying the plasmid are selected and
grown up. As they reproduce, they replicate the plasmid and pass it on to their offspring, making copies of
the DNA it contains.

What is the point of making many copies of a DNA sequence in a plasmid? In some cases, we need lots of
DNA copies to conduct experiments or build new plasmids. In other cases, the piece of DNA encodes a
useful protein, and the bacteria are used as “factories” to make the protein. For instance, the human insulin
gene is expressed in E. coli bacteria to make insulin used by diabetics.

Applications Of Gene Cloning

Listed below are the applications of gene cloning:

• Gene Cloning plays an important role in the medicinal field. It is used in the production of hormones,
vitamins and antibiotics.
• Gene cloning finds its applications in the agricultural field. Nitrogen fixation is carried out by
cyanobacteria wherein desired genes can be used to enhance the productivity of crops and
improvement of health. This practice reduces the use of fertilizers hence chemical-free produce is
generated.
• It can be applied to the science of identifying and detecting a clone containing a particular gene
which can be manipulated by growing in a controlled environment.
• It is used in gene therapy where a faulty gene is replaced by the insertion of a healthy gene. Medical
ailments such as leukemia and sickle cell anemia can be treated with this principle.