RECOMBINANT DNA

TECHNOLOGY

GENETIC ENGINEERING
TECHNOLOGY THAT MANIPULATES OR
MODIFIES THE GENE OF AN ORGANISM,
WITH THE INTENTION OF CREATING
IMPROVED PRODUCTS OR METHODS.
TRANSGENIC PLANT/ANIMAL

 MAINLY USES TECHNIQUES OF DNA

RECOMBINANT.
A PROCESS THAT OCCURS RANDOMLY
AND SPONTANEOUSLY IN NATURE.
BUT NOW HAS BEEN MANIPULATED BY
GENETICISTS.

DNA RECOMBINANT OCCURS

NATURALLY THROUGH:
a) Crossing over during meiosis I
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)

TOOLS USED IN GENETIC

ENGINEERING
1.
2.
3.
4.

RE
VECTORS
DNA LIGASE
HOST O/M

RESTRICTION
ENDONUCLEASES/ ENZYMES
Recombinant DNA technology developed from the
discovery of certain bacterial strains which seemed
immune to attack by bacteriophages.
These strains produced special enzymes that
restricted the replication of foreign DNA which had
infected the bacterial cells. (hence they were called
restriction enzyme)
The site at which a restriction enzyme acts is called
the restriction site. This site is typically 4-6
nucleotides long.

Types of recognition sequences

Blunt ended
DNA
Produced
staggered
cuts,
sticky
ends.
Restriction
site which
produces
sticky
ended DNA
after
cleavage palindromi

Sticky
ended DNA

 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

EXAMPLES OF RESTRICTION
ENZYMES AND RESTRICTION SITES

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.

VECTORS
DNA molecules, derived from a plasmid or
bacteriophage into which DNA fragments may be
inserted.
A. Definition - Bacteriophage (phage) are obligate
intracellular parasites that multiply inside bacteria
by making use of some or all of the host
biosynthetic machinery (i.e., viruses that infect
bacteria.).
A 'plasmid' is a DNA molecule separate from the
chromosomal DNA and capable of autonomous
replication. In many cases, It is typically circular and
double-stranded. It usually occurs in bacteria, and is
sometimes found in eukaryotic organisms (e.g., the
2-micrometre-ring in Saccharomyces cerevisiae).

This vector can then carry the foreign DNA

Properties of Plasmids
1. 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.

Examples of some vectors used in

genetic engineering

pBR322
pUC18
YAK
lambda phage

VECTORS

Cloning Vector

322..

Expression vectors

Expression Vectors

Bacteria Artificial Chromosome

(BACs)

Yeast Artificial Chromosomes

(YACs)

Ti vectors
- Naturally occuring plasmids (around 200 kb
in size
- -isolated from bacterium Agrobacterium
tumefaciens, which is a soil borne plant
pathogen that causes disease namely as
crown gall disease.

DNA LIGASE
Required to anneal or join the sugarphosphate
backbones
in
the
recombinant DNA molecule.

HOST O/M

Vectors unable to replicate independently

(outside the cell)

So they have to be taken up by a host

o/m.

E. coli is the most widely used host

organism :

1) as its biological and genetic characteristics

are well known
2) Produces rapidly
) Other host o/m yeast and mammalian
cells.

CLONING
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.

CLONING

FIVE BASICS STEP IN CLONING PROCESS:

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.

Screening of cell clones carrying the gene

interest

Example :
The process of insulin production by
E. coli as an example.
Note that the chances of success
are very small !!!

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 confers resistance to the
antibiotic ampicillin. Thus bacteria with
this plasmid can grow in media which
contain ampicillin.

The lac Z gene codes for the

synthesis of B-galactosidase which
hydrolyses lactose.
The plasmid has a single restriction
site recognised by the restricion
endonuclease, and the site is
situated in the lac Z gene.

STEP 2 (Recombination)
Half of the RE solution is mixed with the
human DNA
This will produce several hundred
thousand DNA fragments with sticky ends.
The rest of RE enzyme is then mixed with
the plasmid molecules and this will
produce an equally large number of linear
plasmids molecules with sticky ends
complementary to those of the target DNA
fragments.

 The human DNA fragments are mixed with

the plasmid fragments ATP and 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.

STEP 3 (TRANSFORMATION)
The mixture of DNA molecules produced
in step 2 is added to a culture of bacterial
cells, usually E coli.
Calcium ions are added ---stimulated to
take up small pieces of DNA
Only about 1% -----recombinant DNA.

STEP 4 (CLONING)
The bacteria are plated onto nutrient
plates which contain ampicilin and
sugar called X-gal.
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.
P? Q? R?

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.

B-Galactosidesidase hydrolyses X-gal and

produce a blue compound, so bacteria
colonies with functional lac Z gene will be
blue.

 Bacteria colonies with

recombinant
plasmids will form white colonies.
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.

STEP 5 (SCREENING)
A nitrocellulose membrane or filter paper is
laid on the surface of a nutrient agar plate
on which the bacteria colonies with
recombinant
plasdmids
have
been
cultured.
Bacteria cell will be transferred to the filter.
The filter is treated with sodium hydroxide
solution which denatures the DNA by
breaking the hydrogen bonds between the
two polynucleotide strands.
This leaves single-stranded DNA on the
filter. Single-stranded DNA will pair up
(hybridise) with any nucleic acid molecule
with a complementary sequene of bases.

 The filter is incubate with a solution containing gene

probe molecules. The gene probe for insulin is easily
obtained by synthesising a short sequene of
nucleotides which corresponds to the sequence of
animo acids in insulin.
The radioisotope 32p is used in this process so that
the gene probe is radioactive.
Wherever the gene probe meets a complementary DNA
sequence on the nitrocellulose filter, it will bonds with it,
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.