Course: B.SC.

Botany
Semester: IV
Paper Code: BOT CC-408
Paper Name: Molecular Biology
Topic: Recombinant DNA Technology
Faculty Name: Dr.Anjana Verma
Department : Botany
Email Id: [email protected]

RECOMBINANT DNA TECHNOLOGY

Definition
Recombinant DNA Technology is defined as the technique used to create
new combination of genetic material by joining DNA molecules from different organisms.
The process involves cutting and joining of DNA at specific sites in order to produce
chimerical DNA which can be expressed in the new host.
Biotechnology which is synonymous with Genetic Engineering or
Recombinant DNA Technology (rDNA) is an industrial process that uses the
scientific research on DNA for practical applications. Recombinant (rDNA) that is made
through the combination or insertion of one or more DNA strands. Recombinant DNA
Technology deals in constructing new combination of genes in laboratory.
Genetic Engineering is used for creating multiple copies of genes and
insertion of foreign genes into an organisms to give them new traits.

Introduction
Recombinant DNA Technology or Gene Cloning is a new born discipline of
science which aims to alter the heredity of a living organism.
Recombinant DNA is a form of artificial DNA which is made through the
combination or insertion of one or more DNA strands therefore combining DNA
sequences.
Genetic Engineering aims at manipulating the genes of an organism at will
where techniques are applied on the biological system for the benefit of mankind.

Basic Principles of RDT

1 Generation of DNA fragments and selection of the desired piece of DNA.
2 Insertion of selected DNA into a cloning vector to create a recombinant vector.
3 Introduction of the recombinant vector into a host cell
4 Multiplication and selection of clones containing the recombinant molecule.
5 Expression of the foreign gene into the host to produce the desired product.
Steps of Recombinant DNA Technology

1 Isolation/Synthesis of gene
2 Selection of a vector
3 Attachment of foreign gene to the vehicle
4 Transfer of recombinant DNA to the host
5 Expression of the transferred genome

Fig. The steps for producing Recombinant DNA

STEP 1 ISOLATION OF GENE

Identification and isolation of the desired gene is required in order to

generate new combination of genome. Significant progress has been made in the
techniques for isolation of a variety of genes like rRNA , protein products, regulatory
genes, promoter genes, etc. The first gene to be isolated was the lac- operon of
E.coli (Shapiro et al , 1969)
 Artificial ( Organochemical ) synthesis of gene
It was definitely a positive step of genetic engineering. H.G.Khorana (1970) was
successful in synthesizing a double stranded DNA corresponding to the major Yeast
alanyl tRNA.

STEP 2 SELECTION OF VECTOR

Vectors are the DNA molecules which can work as vehicle to carry foreign
gene, either isolated or synthesized, into the host for multiplication or expression.
Vectors are selected according to the purpose and type of host in which it
has to be cloned or expressed named as shuttle vector, cloning vector etc
Some common vectors are

1. Plasmids for prokaryotic host

2. Phages ( Lambda phage, M13 phage)
3. Yeast plasmid for eukaryotic host
4. Ti and Ri plasmid for plants
5. Phagemids ( pUC118, pUS119)
6. Transposons ( P element of Drosophila, Ac & Ds of maize)Yeast Artificial
Chromosome) & Mammalian Artificial Chromosome (YAC & MAC)
7. Artificial Plasmids ( pBR322 , pBR327).
 Fig: Plasmid being used as vector for gene transfer

STEP 3 Attachment of foreign gene to the vehicle

To produce recombinant DNA, site specific cutting and joining is the most
important step. For cutting a duplex DNA at recognized site various Restriction
enzymes (RE) has been isolated. Most suitable amongst them are Type II
Restriction Enzyme which breaks the polynucleotide chain within recognized
sequence for ex- Eco RI , Hae III, etc.
 Restriction enzyme recognize a specific sequence of nucleotides and produce
a double-stranded cut in the DNA. The recognition site is usually 4 to 8 bases.
Recognition site

A palindromic recognition site reads the same on the reverse strand as it does
on the forward strand when both are read in the same orientation.

EcoRI digestion produces sticky ends

Derived from Escherichia coli

Where as SmaI restriction cleavage produces blunt ends.:

Derived from Serratia marcescens

Different restriction enzymes produces DNA strands having different lengths of

nucleotides but having same base pairs at the ends. The DNA generally are
modified or methylated at restriction sites to protect themselves from own restriction
endonucleases. On the basis of recognition site and modification sites RE are
classified
Vector/ Vehicle and Passenger (Foreign) DNA can be joined together to
produce heterologous DNA with the help of Ligase enzyme.

Fig. Cutting of duplex DNA with Eco RI to produce sticky ends (staggered cut)
STEP 4 Transfer of recombinant DNA to the host
After generation of recombinant DNA it is transferred into a suitable host
either for multiplication (cloning) or for expression. Blackman et al (1977) have
conducted a shot gun experiment to obtain an adequate number of E.coli clones
carrying random fragments of Yeast DNA.
A rapid and simple technique for introducing cloned genes into a wide
variety of microbial, plant and animal cell is by Electroporation were high voltage
electric pulses can increase capability of plasma membrane to take in foreign
molecules.
Transduction and Transfection are methods commonly used for
transferring genetic material into bacterial/animal/plant cells.

Fig: Transfer of gene into a bacterial host

 STEP 5 Expression of the transferred genome
The control of replication and expression of the cloned DNA is one of the
most important aspects of beneficial utilization of the Recombinant DNA Technology.
The finding that the rat insulin gene can be transcribed and translated in E.coli cells
open the possibility of large scale and economic production of insulin which is used as
medicine for diabetic patients.
There are various applications of RDT like large scale production of
alcohol, ascetic acid, enzymes , flavoring agents, etc.
Gene therapy, DNA fingerprinting, Diagnosis of molecular diseases are
milestones of RDT.
In the field of Agriculture Tansgenic plants has increased tremendously
the yield and quality of cereals, fruits and vegetables. BT brinjal, Golden rice,
Delayed ripening tomato, Pomato , etc are few examples.

ROLE OF RDT IN HUMAN WELFARE

Recombinant DNA Technology has been applied in different fields for the welfare of
mankind. Following are some of its applications:

1. Application in Crop Improvement

Genetic Engineering has several potential applications in crop improvement.
a. Distant Hybridization -- Due to advancement in GE it is now possible to
transfer genes between distantly related species even genus, for example
Raphanobrassica, Triticale, Pomato, etc.
b. Development of Transgenic plants -- Genetically transformed plants which
contain foreign genes are called transgenic plants. Resistance to disease,
insects and pests, herbicides, drought, metal toxicity tolerance, etc can be
achieved through this Technology. Some of the successful results are BT
Brinjal, BT Cotton, Delayed ripening Tomato, etc.
c. Development of Root Nodules in Cereal Crops -- The bacterial gene
responsible for nitrogen fixation can be transferred now to cereal crops like
wheat, maize, rice, barley etc which enables them to fix nitrogen like
leguminous plants.
d. Grains with Improved Nutritional Characteristics -- Rice grains do not
contain carotene but Genetic engineering has led to develop a rice plant
(Golden Rice) which produce yellow grains because of high carotene
content. About 300 grams of this cooked rice a day can supply all the α-
carotene a person needs.
2. Application in Medicine
Genetic Engineering plays an important role in the field of Medical Science
a. Production of Antibiotics -- Penicillium and Streptomyces fungi has
been used for mass production of famous antibiotic Penicillin and
Streptomycin. Genetically efficient strains of these fungi have been developed
to greatly increase the yield of these antibiotics.
b. Production of Insulin -- Insulin is a hormone, used by diabetics, was
usually extracted from pancreas of cows and pigs. This insulin was slightly
different in structure from human insulin. As a result, it leads to allergic
reactions. It was expensive also. By using RDT Human gene for insulin
production has been incorporated into bacterial DNA and such genetically
engineered bacteria are used for large scale production of insulin. This insulin
does not cause allergy. This has reduced the cost price of insulin as well.
c. Production of Vaccines --- A vaccine is a substance containing all or
some part of a harmless version of pathogen that is introduced into the body
to produce immunity against the pathogen. A DNA vaccine is a vaccine made
from the DNA of a pathogen but does not have disease causing capability.
This DNA vaccine is injected into a patient where it directs the synthesis of
protein. The immunity develops against the protein. Researchers are working
to develop DNA vaccines to prevent AIDS, malaria and cancers.
d. Plantibodies -- An antibody produced by genetically modified crops are
known as plantibodies. Plants are transformed by introducing antibody genes
from animals. This was done in 1989, with a mouse antibody made by
tobacco plant. Some successful results are -- Hepatitis B vaccine, vaccine
against HIV virus, Anthrax vaccine from tobacco plants (one acre of plant can
produce 360 million doses in a year ).
e. Gene Therapy -- Genetic Engineering may one day enable the medical
Scientists to replace the defective genes responsible for hereditary diseases
like Haemophilia, Phnylkeptonuria, etc with normal genes.
f. Production of Enzymes -- Some useful enzymes can be produced by
Recombinant DNA Technology. The conversion of plasminogen to plasmin is
activated by an enzyme called Tissue Plasminogen Activator (TPA), which
is produced by lining of blood vessels. Heart attacks and many strokes are
caused by blood clots. Treating these persons with this enzyme saved lifes.
The discovery of TPA and its isolation from human tissues was used to make
recombinant plasmid inserted into an expression vector, was transfected into
E.coli. The transgenic bacteria made the protein in quantity and it soon
became commercially available.

The following table shows some medically useful recombinant products and their
applications:
3. Industrial Application
In industries, RDT helps in the production of chemical compounds of
commercial importance. Improvement of existing fermentation processes and
production of proteins from wastes. This can be achieved by developing more
efficient strains of microorganisms. This is also known as crop improvement
which ultimately increase the production of chemicals, medicines, enzymes, etc.

4. Generation of Transgenic Animals

The gene encoding the growth hormone somatotropin was first to be
cloned successfully. The dairy farmers use Bovine Somatotropin (BST) worldwide
as a supplement to their cows diets, increasing the animals milk production.
5. Application in Forensics
Genetic Engineering has an enormous impact on the field of forensic
science. PCR (Polymerase Chain Reaction) or RFLP (Restriction Fragment
Length Polymorphism) analysis can be used in criminal investigations. Both
techniques produce a fingerprint or pattern of bands on gel. They use the non-
coding regions of the human genome which can be used as a basis for
discriminating between individuals.

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