NIT PATNA

 Recombinant DNA technology is 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 technology has made it

possible to isolate one gene or any other
segment of DNA, enabling researchers to
determine its nucleotide sequence, study its
transcripts, mutate it in highly specific ways,
and reinsert the modified sequence into a
living organism.
 Recombinant DNA is possible because DNA molecules
from all organisms share the same chemical structure.
They differ only in the nucleotide sequence within that
identical overall structure.
 The DNA sequences used in the construction of
recombinant DNA molecules can originate from
any species. For example, plant DNA may be joined to
bacterial DNA, or human DNA may be joined with fungal
DNA.
 In addition, DNA sequences that do not occur anywhere
in nature may be created by the chemical synthesis of
DNA, and incorporated into recombinant molecules.
Using recombinant DNA technology and synthetic DNA,
literally any DNA sequence may be created and
introduced into any of a very wide range of living
organisms.
 Step 1: Identification and isolation of gene of
interest.
 Step 2 : Insertion of isolated gene in a suitable
vector.
 Step 3 : Transfer of rDNA molecules in an
appropriate host cell. The rDNA molecules are
generated when the vector itself replicates in the
host cell.
 Step 4 : Selection and separation of transformed
host cell.
 Step 5 : Replication of cell containing rDNA
molecules to get a genetically identical cells
population or clone.
Here two steps are involved in isolation of DNA :
 Cutting DNA
 Joining DNA

Restriction enzymes(or restriction endonucleases)

are used to cut DNA.It recognizes one
particular nucleotide sequence in DNA and
cuts the DNA molecule (breaks down the bond
between two nucleotides) .
 Like all enzymes, a restriction
enzyme works by shape-to-
shape matching. When it
comes into contact with a DNA
sequence with a shape that
matches a part of the enzyme,
called the recognition site, it
wraps around the DNA and
causes a break in both strands
of the DNA molecule.
 Each
restriction enzyme recognises a
different and
specific recognition site, or DNA
sequence. Recognition sites are
usually only short - 4-
8 nucleotides.
e.g. In the above case, the restriction enzyme EcoRI
cuts the circular DNA molecule bearing target
sequence resulting in linear molecule with single
stranded sticky ends.
 The cut DNA fragments
are covalently joined
by DNA ligases.
 DNA ligase join the DNA
fragments by forming a
phosphodiester bond
between the
phosphate group of
5’-carbon of one
deoxyribose with the
hydroxyl group of 3’-
carbon of another
deoxyribose.
 A vector is a DNA
molecule which is
capable of
multiplying inside the
host to which our
gene of interest is
integrated for cloning.
 Ligase is the joining
enzyme which joins
the vector DNA with
gene of interest.
 Small pieces of DNA used for cloning (the gene to be
inserted into the genetically modified organism must be
combined with other genetic elements in order for it to work
properly).
 Requirements of the Vector
1. Self-replication - able to replicate in the host (origin of
replication)
2. Cloning site - site for recognition of restriction nucleases.
3. Promoter (and operator) - to support the gene (new DNA)
expression in the host
4. Selectable marker - antibiotic resistance
5. Proper size
 Commonly used vectors
are :
 Plasmids-Plasmids are self-
replicating circular molecules of
DNA
 Bacteriophages
 Cosmids
 Artificial chromosome
vectors
Commonly used host cells
are :
 Bacteria
Fig: Plasmid inside E.Coli
 Yeasts
 Plant cells
 Mammalian cells
There are many methods of inserting DNA into host cell:
 Transformation
 Electroporation
 Protoplast fusion
 Microinjection

 The vector is added to a flask containing E-coli.

 CaCl2 is added to the flask followed by brief heat shock.
 This allows the holes to appear in the cell membrane of E-
coli making it permeable to DNA and allowing the plasmids
to enter.
 Here the plasmids with the gene of interest attached to it
replicates.
 After step 3, we get three types of E-coli bacterial
cell:

We only have to focus on transformed bacterial cell with

recombinant vector.
 Antibiotic solution is
added to the
transformed host cell.
 The E-Coli bacterial cell
which have plasmids
containing gene of
interest and antibiotic
resistance gene are
remains while other Antibiotic
bacterial cells are killed
by the antibiotic solution.
 Then the rest of the cells
are allowed to replicate
 The Plasmids
replicate inside the
E.Coli cell and
hence a large
number of
genetically modified
E.Coli are produced
containing rDNA
genes.
 Gene Therapy:
This is achieved by cloning a gene into a vector that will
readily will be taken up and incorporated into the genome of
the host cell.
 Agriculture:
Genetically engineered plants are developed to resist
draught and diseases. Good quality foods and increased
yield of crops is also possible.
 Industrial application:
Enzymes used to produce sugar, cheese, detergents. Protein
products used as food additives, increases nutritive value,
besides imparting flavour.
 Application in forensic medicine:
DNA Fingerprinting helps to identify criminals and settle
disputes of parenthood of children.
 Gene Cloning :
The recombinant DNA is transferred to host cell. Within the host
cell it replicates creation dozens of identical copies.
 An experimental technique for correcting defective genes that are
responsible for disease development.
 The most common form of gene therapy involved inserting a normal
gene to replace an abnormal gene.

 Approaches used:
1. Replacing a mutated gene that causes diseases with a healthy copy of
the gene.
2. .Inactivating or knocking out , a mutated gene that is functioning
improperly.
3. Introducing a new gene into the body to help fight a disease.

Researches are studying gene therapy for a number of diseases, such as:
 Severe combined immuno-deficiencies(SCID)
 Hemophilia
 Parkinson's disease
 Cancer
 HIV