Genetic Engineering

and Applications of
Recombinant DNA
GENERAL BIOLOGY 2
 Learning Objectives:

1 2 3 4
compare classical enumerate the steps describe some explain the selection
breeding with in molecular cloning; methods to and screening of
modern genetic introduce DNA into transformants /
engineering cells; and genetically modified
techniques; organisms (GMOs).
 The procedure involves:
• Selection and Isolation of DNA
Steps • Cutting the gene at the recognition
involved in sites.
• Amplifying the gene copies through
Recombinant Polymerase chain reaction (PCR).
DNA • Selection of suitable cloning vector
• Ligation of DNA Molecules. (DNA
Technology Ligase)
• Insertion of Recombinant DNA Into
Host.
Key Terms:
Gene- the basic physical and functional unit of heredity

Plasmid-These are double stranded DNA that are usually circular and mostly found inside
certain bacterial specie e.g. E.coli
Restriction Enzyme- also called restriction endonucleases a protein isolated from bacteria
that cleaves DNA sequences at sequence-specific sites, producing DNA fragments with a
known sequence at each end.

DNA Ligase- a DNA-joining enzyme

Vectors- are genetic sequences derived from viruses or bacteria.

Host Organism- into which the recombinant DNA is introduced.

1. Selection and
Isolation of DNA
insert
▪ A gene of interest is first
identified and isolated from
the thousands of genes an
organisms may have.
2. Cutting DNA at
precise locations
▪ The isolated gene is then cut from its
source DNA molecule with
restriction enzymes.
 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 restriction
enzymes.
 4. Selection of
suitable cloning
vector
▪ A cloning vector is a self-
replicating DNA molecule, into
which the DNA insert is to be
integrated. A suitable cloning
vector is selected in the next step
of rec DNA technology. Most
commonly used vectors are
plasmids and bacteriophages.
 5. Ligation of DNA
Molecules

▪ The target DNA or the DNA insert

which has been extracted and cleaved
enzymatically by the selective
restriction endonuclease enzymes are
now ligated (joined) by the enzyme
ligase to vector DNA to form a rDNA
molecule which is often called as
cloning-vector-insert DNA construct.
 6. Insertion of
Recombinant DNA Into
Host

▪ Suitable host cells are selected and the

rDNA molecule that is formed is
introduced into these host cells. This
process of entry of rDNA into the host
cell is called transformation. Usually
selected hosts are bacterial cells like E.
coli, however yeast, fungi may also be
utilized.
 7. Expression and
Multiplication of DNA
insert in the host:

▪ Finally, it is to be ensured that the

foreign DNA inserted into the vector
DNA is expressing the desired
character in the host cells. Also, the
transformed host cells are multiplied
to obtain sufficient number of copies.
If needed, such genes may also be
transferred and expressed into another
organism
 Mating two members of a species (plant,
yeast, or animal)—each of whom
possesses one or more different and
desirable traits—in order to create a
Classical hybrid individual possessing both traits.

Breeding Uses deliberate interbreeding (crossing)

of closely or distantly related individuals
to produce new crop varieties or lines
with desirable properties.
APPLICATIONS OF
RECOMBINANT DNA
PCR Amplification
• A technique that allows the detection of specific genes in
target organisms.
• PCR amplification is an in-vitro method that simulates DNA
replication in vivo. It utilizes a thermostable (heat-resistant)
DNA polymerase that builds single stranded DNA strands
unto unwound DNA templates.