In Vitro Genetic Manipulation or

Recombinant DNA Technology

Recombinant DNA Technology
the use of in-vitro techniques in the isolation,
manipulation, recombination and expression of
DNA

Four Basic Requirements for DNA

Technology
1. Vector
2. Method of splicing foreign genetic
material into the vector
3. Method of introducing the vector/foreign
DNA recombinants into the host cell
4. Method of Assay

1. Vector
Has the capacity to enter a host cell
and replicate within and be
maintained in it
e.g
Plasmids
Cosmids
BACS (Bacterial Artificial Chromosome)

Plasmids
an extra-chromosomal DNA molecule
separate from the chromosomal DNA
which is capable of replicating
independently of the chromosomal
DNA
able to contain 1-20 kbp of DNA

2. Method of splicing foreign genetic

material into the vector
Made use of the discovery of site specific
Restriction Enzyme (RE)
Some RE cut straight across the double
helix producing "blunt" ends. However,
many restriction enzymes cut in an offset
fashion. The ends of the cut have an
overhanging piece of single-stranded
DNA. These are called "sticky ends"

Restriction enzymes
(Molecular Scissors)
DNA-cutting enzymes found in
bacteria
recognizes and cuts DNA only at a
particular sequence of nucleotides.
(restriction site)
e.g., HaeIII (Hemophilus
aegypticus)
5'GGCC3'

RE

RE

Method of introducing the

vector/foreign DNA recombinants into
the host cell
1. Transformation
2. Electroporation
3. Heat-shock

Method of introducing the

vector/foreign DNA recombinants
into the host cell
1. Transformation
. Transfer of cell-free DNA or exogenous
DNA from one strain to another
Need cells to be competent
certain stage of growth
Induced = CaCl2

Method of introducing the

vector/foreign DNA recombinants into
the host cell
2. Electroporation
transfer of DNA is brought about
by the application of pulsed field to
the cells

2. Electroporation

electroporator

Method of Assay
(Screening)
Assay for the foreign gene product of
choice from the population of
recombinant clones
Ex. Ampicillin-resistance that is
activated when gene of interest
is connected to vector

Method of Assay (Blue and White

Screening)

Uses of Recombinant DNA

Technology
1. Increase enzyme production
2. Creation of novel products
e.g hormones, interferon
3. Increase knowledge of genetics at
the molecular level

Practical Applications of
Recombinant DNA Technology
Pharmaceutical Products
a. E. coli
insulin
growth hormones,
inteferons

Practical Applications of
Recombinant DNA Technology
2.Production of Specific Biodegrading
Organisms for Pollution Control
Ex. Oil-degrading bacteria