Recombinant DNA technology

The technology used for producing artificial DNA through the combination of different genetic materials (DNA) from
different sources is referred to as Recombinant DNA Technology. Recombinant DNA technology is popularly known
as genetic engineering.
Recombinant DNA technology is defined as the development and application of scientific methods, procedures, and
technologies that permit direct manipulation of genetic material in order to alter the hereditary traits of a cell,
organism, or population.
Recombinant DNA refers to the creation of new combinations of DNA segments that are not found together in nature.
The isolation and manipulation of genes allows for more precise genetic analysis as well as practical applications in
medicine, agriculture, and industry.
How recombinant DNA technology work?
• The organism under study, which will be used to donate DNA for the analysis, is called the donor organism.
• The basic procedure is to extract and cut up DNA from a donor genome into fragments containing from one to
several genes and allow these fragments to insert themselves individually into opened-up small autonomously
replicating DNA molecules such as bacterial plasmids.
• These small circular molecules act as carriers, or vectors, for the DNA fragments. The vector molecules with their
inserts are called recombinant DNA because they consist of novel combinations of DNA from the donor genome
(which can be from any organism) with vector DNA from a completely different source (generally a
bacterial plasmid or a virus).
• The recombinant DNA mixture is then used to transform bacterial cells, and it is common for single recombinant
vector molecules to find their way into individual bacterial cells.
• Bacterial cells are plated and allowed to grow into colonies. An individual transformed cell with a single
recombinant vector will divide into a colony with millions of cells, all carrying the same recombinant vector.
• Therefore, an individual colony contains a very large population of identical DNA inserts, and this population is
called a DNA clone.
TOOLS OF RECOMBINANT DNA TECHNOLOGY
Enzymes: Restriction enzymes help to cut. the polymerases help to synthesize and the ligases help to bind. The
restriction enzymes determine 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 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 (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 possess an origin of replication (sequence of nucleotide
from where the replication starts), a selectable marker (genes which show resistance to certain antibiotics); 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. Using one of the
several methods these recombinant DNAs are inserted into the host, namely – microinjection, biolistics or gene gun,
alternate cooling and heating, use of calcium ions, etc.
STEPS OF RECOMBINANT DNA TECHNOLOGY: The complete process of recombinant DNA
technology includes multiple steps, maintained in a specific sequence to generate the desired product.
Step-1. Isolation of Genetic Material: The first and the initial step in Recombinant DNA technology is to isolate the
desired DNA in its pure form i.e. free from other macromolecules.
Step-2. Cutting the gene at the recognition sites: The restriction enzymes play a major role in determining the
location at which the desired gene is inserted into the vector genome. These reactions are called ‘restriction enzyme
digestions.
Step-3. Amplifying the gene copies through Polymerase chain reaction (PCR): It is a process to amplify a single
copy of DNA into thousands to millions of copies once the proper gene of interest has been cut using the restriction
enzymes.
Step-4. Ligation of DNA Molecules: In this step of Ligation, joining of the two pieces – a cut fragment of DNA and
the vector together with the help of the enzyme DNA ligase.
Step-5. Insertion of Recombinant DNA Into Host and Amplifying recombinant DNA: In this step, the
recombinant DNA is introduced into a recipient host cell. This process is termed as Transformation. Once after the
insertion of the recombinant DNA into the host cell, it gets multiplied and is expressed in the form of the
manufactured protein under optimal conditions. As mentioned in Tools of recombinant DNA technology, there are
various ways in which this can be achieved. The effectively transformed cells/organisms carry forward the
recombinant gene to the offspring.
Transformation*: In transformation, pieces of DNA released from donor bacteria are taken up directly from the
extracellular environment by recipient bacteria. Recombination occurs between single molecules of transforming
DNA and the chromosomes of recipient bacteria. To be active in transformation, DNA molecules must be at least 500
nucleotides in length, and transforming activity is destroyed rapidly by treating DNA with deoxyribonuclease.

Applications of Recombinant DNA Technology in medicine: Genetic engineering or rec DNA technology has
enormous and wide-spread applications in all the fields of biological sciences. Some important applications of rec
DNA technology in medicine are enlisted below:
(1) Production of Transgenic Medicinal Plants: By utilizing genetic engineering it is possible to produce transgenic
plants or the genetically modified plants. Many transgenic plants have been developed with better qualities like
resistance to herbicides, insects or viruses or with expression of male sterility, etc. Also, they allow the production of
commercially important biochemical, pharmaceutical compounds, etc.
(2) Production of Transgenic Animals: using rec DNA technology, desired genes can be inserted into the animal to
produce the transgenic animal. It helps for the production of better farm animals to ensure more commercial benefits.
Commercially important use of transgenic animals is the production of certain proteins and pharmaceutical
compounds. Transgenic animals also contribute for studying the gene functions in different animal species.
(3) Production of Hormones: bacterial cells like E. coli are utilized for the production of different fine chemicals like
insulin, somatostatin, somatotropin and p-endorphin. Human Insulin Hormone i.e., Humulin is the first therapeutic
product which was produced by the application of rec DNA technology. The genes of interest are incorporated into the
bacterial cells which are then cloned. Such clones are capable of producing a fair quantity of hormones like insulin
which have great commercial importance.
(4) Production of Vaccines: Vaccines contains a pathogen in attenuated (or weakened) or inactive state that may be
given to human beings or animals to confer immunity to infection. A number of vaccines have been synthesized
biologically through rec DNA technology. These vaccines are effective against numerous serious diseases caused by
bacteria, viruses or protozoa. These include vaccines for polio, malaria, cholera, hepatitis, rabies, smallpox, etc. DNA-
vaccine is the preparation that contains a gene encoding an immunogenic protein from the concerned pathogen.
(5) Biosynthesis of Interferon: Interferon’s are the glycoproteins which are produced in very minute amounts by the
virus-infected cells. Interferon’s have antiviral and even anti-cancerous properties. By rec DNA technology method,
the gene of human fibroblasts (which produce interferon’s in human beings) is inserted into the bacterial plasmid.
These genetically engineered bacteria are cloned and cultured so that the gene is expressed and the interferon’s are
produced in fairly high quantities. This interferon, so produced, is then extracted and purified.
(6) Production of Antibiotics: Antibiotics produced by microorganisms are very effective against different viral,
bacterial or protozoan diseases. Some important antibiotics are tetracyclin, penicillin, streptomycin, novobiocin,
bacitracin, etc. rec DNA technology helps in increasing the production of antibiotics by improving the microbial
strains through modification of genetic characteristics.
(7) Production of Commercially Important Chemicals: Various commercially important chemicals can be
produced more efficiently by utilizing the methods of rec DNA technology. A few of them are the alcohols and
alcoholic beverages obtained through fermentation; organic acids like citric acid, acetic acid, etc. and vitamins
produced by microorganisms.
(8) Application in Enzyme Engineering: As we know that the enzymes are encoded by genes, so if there are changes
in a gene then definitely the enzyme structure also changes. Enzyme engineering utilizes the same fact and can be
explained as the modification of an enzyme structure by inducing alterations in the genes which encode for that
particular enzyme.
(9) Prevention and Diagnosis of Diseases: Genetic engineering methods and techniques have greatly solved the
problem of conventional methods for diagnosis of diseases. It also provides methods for the. prevention of a number
of diseases like AIDS, cholera, etc. Monoclonal antibodies are useful tools for disease diagnosis. Monoclonal
antibodies are produced by using the technique called hybridoma technology. The monoclonal antibodies bear
specificity against a specific antigen. These are used in the diagnosis of diseases due to their specificity. Genetic
engineering allows the production of hybridoma which is a cell obtained by the fusion of a lymphocyte cell capable of
producing antibodies and a single myeloma cell (tumor cell).
(10) Gene Therapy: Gene therapy is undoubtedly the most beneficial area of genetic engineering for human beings. It
involves delivery of specific genes into human body to correct the diseases. Thus, it is the treatment of diseases by
transfer and expression of a gene into the patients’ cells so as to ensure the restoration of a normal cellular activity.
(11) Practical Applications of Genetic Engineering: rec DNA technology has an immense scope in Research and
Experimental studies. It is applied for:
a. Localizing specific genes. d. Molecular analysis of various diseases.
b. Sequencing of DNA or genes. e. Study of” mutations in DNA, etc.
c. Study of mechanism of gene regulation.
(12) Applications in forensic science: DNA profiling or DNA fingerprinting, enables us to identify any person by
his hair roots Wood stains, serum, etc. it also helps to solve the problems of parentage and to identify the criminals.