GENETIC ENGINEERING
(RECOMBINANT DNA
TECHNOLOGY)
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 GENETIC ENGINEERING:
 technology that manipulates/ modifies the gene
of an organism, with the intention of creating
improved products or methods, such as
transgenic plant/animal.
 mainly uses techniques of DNA recombinant.
 DNA recombinant is a process that occurs
randomly and spontaneously in nature.
 but now it has been manipulated by geneticists
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DNA RECOMBINANT OCCURS
NATURALLY THROUGH:
Crossing over during meiosis
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b) Sexual recombination when
chromosomes from two
separate individuals combine
to produce offspring.
c) Spontaneous mutations
d) Bacterial transformations
e) Viral infections
- Random and undirected (no
specific goal)
a)
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Gene Cloning: isolation and
making of many copies of a gene
Cloning in recombinant technology is defined as
the production of a cell line or culture, all of
whose members contain identical copies of
particular nucleotide sequence.
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TOOLS USED IN GENETIC
ENGINEERING
1. RE (restriction endonuclease/ enzyme)
2. vectors
3. DNA ligase
4. host organism
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Vector
A small piece of DNA where a gene of interest is
introduced (vector acts as a gene carrier).
The vector is then placed within a living cell (host
cell), where it replicates and produces multiple
identical copies of the inserted gene.
One of the most commonly used vectors are
plasmids.
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Plasmids as vector
Plasmids are small circular pieces of DNA that
are found in bacterial and some eukaryotic cells.
Plasmids contain origins of replication that are
recognized by the host cell.
Many plasmids contain selectable markers, can
grow in the presence of an antibiotic or other
toxic substance.
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Vectors unable
(outside the cell)
to
replicate
independently
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Properties of Plasmids
Small molecules with known structures
2. Contain an origin of replication ---the plasmid
can replicate itself in the host cell
3. Have one or more restriction sites recognised
by RE
4. They code for special functions which confer
well-defined features on the host, thus making
them easier to be selected.
1.
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RESTRICTION ENDONUCLEASES/ ENZYMES (RE)
 REs recognise base sequences and then cleave the
DNA at two defined locations .
 The site at which a restriction enzyme acts is called the
restriction site. This site is typically 4-6 nucleotides
long.
 RE is found in many bacterial species, where they
protect the organism from the invasion of foreign DNA
 REs usually recognise palindromic sequences.
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 Some REs produce fragments with “sticky ends,” where they
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will form hydrogen bond with each other due to their
complementary sequences.
This sticky ends are single stranded and they can pair with the
complementary single stranded ends of other DNA molecules
which have been cut with the same enzyme.
Thus possible to bind DNA fragments from different sources.
This is called recombinant DNA
a single restriction enzyme may cut a DNA molecule in several
places because the target sequence usually occurs many
times in a long DNA molecule.
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Types of recognition sequences
•Produced
staggered
cuts, sticky
ends.
•Restriction
site which
produces
sticky ended
DNA after
cleavage palindromic
Blunt ended
DNA
Sticky
ended DNA
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EXAMPLES OF RESTRICTION ENZYMES AND
RESTRICTION SITES
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DNA LIGASE
 Required to anneal or join the sugarphosphate backbones in the recombinant
DNA molecule
 to attach the target DNA (gene of interest)
with vector, becoming recombinant DNA.
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HOST ORGANISM

Vectors (such as plasmids) are unable to
replicate independently (outside the cell)
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So they have to be taken up by a host
organism.

E. coli is the most widely used host organism:
1)
as its biological and genetic characteristics are
well known
2) Produces rapidly
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Other host o/m – yeast and mammalian cells.
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THEBASICS
5 STEPS
OFIN
GENETCI
FIVE
STEP
CLONING EGNINEERING
PROCESS:
USING RECOMBINAT DNA
ISOLATION OF TARGET DNA AND VECTOR DNA
Insertion of target DNA into vector DNA using RE and
DNA ligase
Transformation/transduction of host cells.
- Uptake of recombinant vector DNA by the host cells
Cloning of host cells carrying foreign genesamplification.
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Screening of cell clones carrying the gene
interest
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Example :
 The process of insulin production by E. coli
as an example.
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STEP 1 (ISOLATION)
 Two kinds of DNA are prepared;
1. the gene of interest (insulin gene) which is
cultured from human cells,
2. bacterial plasmid with two particular genes,
that is the ampR gene and the lac Z gene.

The ampR gene give resistance to the
antibiotic ampicillin. Bacteria with this
plasmid can grow in media which contain
ampicillin.
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• The lac Z gene codes for the synthesis of
B-galactosidase which hydrolyses lactose.
The plasmid has a single restriction site
recognised by the restriction endonuclease,
and the site is situated in the lac Z gene.
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STEP 2 (Recombination)
 Half of the RE solution is mixed with target DNA
fragment which produce insulin (target DNA).
 This will produce several hundred thousand DNA
fragments with sticky ends.
 The rest of RE enzyme is then mixed with the
plasmid molecules
 large number of linear plasmid molecules with
sticky ends complementary to the target DNA
fragments will be produced.
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 The target DNA fragments are mixed with the
plasmid fragments, then DNA ligase are added.
 Since all the fragments have complementary
sticky ends, at least three possible combinations
may result from the pairing of these ends.
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STEP 3 (TRANSFORMATION)
 The mixture of DNA molecules produced in
step 2 is added to a culture of bacterial cells
(host), usually E. coli.
 Calcium ions are added to stimulate uptake
of plasmids by host cells.
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STEP 4 (CLONING)
 The bacteria are plated onto nutrient plates
which contain ampicilin and sugar called Xgal.
 Untransformed strains of E. coli will be killed
by ampicilin. Only those cells which have
acquired the plasmid with the ampR gene will
be able to grow in the medium.
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
X-gal in the medium is used to indentify those
colonies of bacteria with recombinant plasmids.

X-gal is hydrolysed by B-galactosidase, an
enzyme which is coded for the lac Z gene. Only
bacteria with intact lac Z genes can produce this
enzyme.
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B-Galactosidesidase hydrolyses X-gal and
produce a blue compound, so bacteria colonies
with functional lac Z gene will be blue.
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Bacteria colonies with recombinant plasmids
will form white colonies.
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STEP 5 (SCREENING)
 Having identified the bacteria colonies with the
recombinant plasmids, the final step is to
determine which of these colonies contain the
DNA sequence which encodes insulin.
 nitrocellulose membrane or filter paper is laid on
the surface of a nutrient agar plate on which the
bacteria colonies with recombinant plasmids
have been cultured.
 Bacteria cell will be transferred to the filter.
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STEP 5 (SCREENING)

The filter is treated with sodium hydroxide (NaOH) 
denatures DNA by breaking the hydrogen bonds
between t 2 polynucleotide strands.
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Single-stranded DNA will pair up (hybridise) with any
nucleic acid molecule with a complementary sequence
of bases.
 The filter is incubated with a solution containing gene
probe molecules.
*gene probe for insulin is easily obtained by synthesizing a short
sequence of nucleotides which corresponds to the sequence of amino
acids in insulin.
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 The radioisotope 32p is used in this process so
that the gene probe is radioactive.
 gene probe will bind to complementary DNA
sequences on the filter, forming a DNA-probe
hybrid.
 The location of these hybrid is determined using
autoradiography and by comparing the
autoradiogram with the original master plate,
those bacteria colonies with the insulin gene are
selected.
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