Recombinant DNA

Technology
BTEC3301
 Recombinant DNA
Technology

Recombinant DNA technology

procedures by which DNA from
different species can be isolated, cut
and spliced together -- new
"recombinant " molecules are then
multiplied in quantity in populations of
rapidly dividing cells (e.g. bacteria,
yeast).
 Recombinant DNA
Technology

The term gene cloning, recombinant

DNA technology and genetic
engineering may seems similar,
however they are different
techniques in Biotechnology and they
are interrelated
 Recombinant DNA
Technology

Human gene therapy, genetically-

engineered crop plants and
transgenicmice have become
possible because of the powerful
techniques developed to manipulate
nucleic acids and proteins.
 Recombinant DNA
Technology

Inthe early 1970s it became possible

to isolate a specific piece of DNA out
of the millions of base pairs in a
typical genome.
 Recombinant DNA
Technology

Currentlyit is relatively easy to cut

out a specific piece of DNA, produce
a large number of copies , determine
its nucleotide sequence, slightly alter
it and then as a final step transfer it
back into cell in.
 Recombinant DNA
Technology

Recombinant DNA technology is based

on a number of important things:
Bacteria contain extrachromosomal
molecules of DNA called plasmids
which are circular.
 Recombinant DNA
Technology

Bacteria also produce enzymes

called restriction endonucleases
that cut DNA molecules at specific
places into many smaller fragments
called restriction fragments.
 Recombinant DNA
Technology
Restriction Enzymes and
plasmid
There are many different kinds of
restriction endonucleases
Each nuclei cuts DNA at a specific
site defined by a sequence of bases
in the DNA called a recognition site
 Recombinant DNA
Technology
Restriction Enzymes and
plasmid
A restriction enzyme cuts only
double-helical segments that contain
a particular sequence, and it makes
its incisions only within that
sequence--known as a "recognition
sequence".
 Recombinant DNA
Technology
Restriction Enzymes and
plasmid
Stickyend and blunt end are the
two possible configurations resulting
from the breaking of double-stranded
DNA
 Recombinant DNA
Technology
Restriction Enzymes and plasmid
If two complementary strands of DNA are of
equal length, then they will terminate in a
blunt end, as in the following example:

5'-CpTpGpApTpCpTpGpApCpTpGpApTpGpCpGpTpApTpGpCpTpApGpT -
3'

3'-GpApCpTpApGpApCpTpGpApCpTpApCpGpCpApTpApCpGpApTpCpA -
5'
 Recombinant DNA
Technology
Restriction Enzymes and
plasmid
However, if one strand extends beyond
the complementary region, then the
DNA is said to possess an overhang:

5'-ApTpCpTpGpApCpT-3'
3'-TpApGpApCpTpGpApCpTpApCpG-5'
 Recombinant DNA
Technology
Restriction Enzymes and
plasmid
Ifanother DNA fragment exists with a
complementary overhang, then these
two overhangs will tend to associate
with each other and each strand is said
to possess a sticky end:
 Recombinant DNA
Technology
Restriction Enzymes and plasmid
5'-ApTpCpTpGpApCpT
pGpApTpGpCpGpTpApTpGpCpT-3'

3'-TpApGpApCpTpGpApCpTpApCpGp
CpApTpApCpGpA-5'

Becomes
5'-ApTpCpTpGpApCpT pGpApTpGpCpGpTpApTpGpCpT-3'

3'-TpApGpApCpTpGpApCpTpApCpGp CpApTpApCpGpA-5'
 Recombinant DNA
Technology
Restriction Enzymes and plasmid
Restriction Enzymes are primarily found
in bacteria and are given abbreviations
based on genus and species of the
bacteria.
One of the first restriction enzymes to
be isolated was from EcoRI
EcoRI is so named because it was
isolated from Escherichia coli strain
called RY13.
 Recombinant DNA
Technology
Digestion of DNA by EcoRI to
Digestion of DNA by EcoRI to
produce cohesive ends ( Fig.
3.1):
 Recombinant DNA
Technology
Creating recombinant DNA :
Creating recombinant DNA :

Thefirst Recombinant DNA

molecules were made by Paul Berg at
Stanford University in 1972.

In
1973 Herbert Boyer and Stanley
Cohen created the first recombinant
DNA organisms.
 Recombinant DNA
Technology
Creating Recombinant DNA (Fig
3.2):
 Recombinant DNA
Technology
Reading materials :Summary of
Recombinant DNA technology process:

Recombinant DNA technology requires DNA

extraction, purification, and fragmentation.

Fragmentation of DNA is done by specific

'restriction' enzymes and is followed by
sorting and isolation of fragments
containing a particular gene.
 Recombinant DNA
Technology
Summary of Recombinant DNA
Summary of Recombinant DNA
technology process:

This
portion of the DNA is then
coupled to a carrier molecule.

Thehybrid DNA is introduced into a

chosen cell for reproduction and
synthesis.
 Recombinant DNA
Technology
Transformation and Antibiotic
Transformation and Antibiotic
Selection

Transformation is the genetic

alteration of a cell resulting from the
introduction, uptake and expression
of foreign DNA.
 Recombinant DNA
Technology
Transformation and Antibiotic
Selection

There are more aggressive techniques for

inserting foreign DNA into eukaryotic cells.
For example, through electroporation.
Electroporation involves applying a brief
(milliseconds) pulse high voltage
electricity to create tiny holes in the
bacterial cell wall that allows DNA to
enter.
 Recombinant DNA
Technology
Plasmids and Antibiotic resistance
Plasmids and Antibiotic resistance
Plasmids were discovered in the
late sixties, and it was quickly
realized that they could be used to
amplify a gene of interest.

A plasmid containing resistance to an

antibiotic (usually ampicillin) or
Tetracycline, is used as a vector.
 Recombinant DNA
Technology
Thegene of interest (resistant to
Ampicillin) is inserted into the vector
plasmid and this newly constructed
plasmid is then put into E. coli that is
sensitive to ampicillin.( Text bk:Pg
58)

Thebacteria are then spread over a

plate that contains ampicillin.
 Recombinant DNA
Technology
Plasmids and Antibiotic resistance
Plasmids and Antibiotic resistance
The ampicillin provides a selective
pressure because only bacteria that
have acquired the plasmid can grow
on the plate.
Those bacteria which do not acquire
the plasmid with the inserted gene of
interest will die.
 Recombinant DNA
Technology
Plasmids and Antibiotic resistance
Plasmids and Antibiotic resistance
Aslong as the bacteria grow in
ampicillin, it will need the plasmid to
survive and it will continually
replicate it, along with the gene of
interest that has been inserted to the
plasmid .
 Recombinant DNA
Technology

Fig 3.3
(a).

Selecting
a Gene in
a plasmid
and
Antibiotic
selection.
 Recombinant DNA
Technology
Assignment: For above
procedure,

Read Transformation of
Bacterial cells and Antibiotic
selection pg 61.
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Technology
Human Gene cloning
Human Gene cloning

Once inside a bacterium, the plasmid

containing the human cDNA can
multiply to yield several dozen
replicas.
Recombinant DNA
Technology
 Recombinant DNA
Technology
Reading materials:
Reading materials:
Summary of Recombinant DNA and
Cloning (Fig. below):

Isolation of two kinds of DNA

Treatment of plasmid and foreign DNA

with the same restriction enzyme

Mixture of foreign DNA with plasmids

 Recombinant DNA
Technology
Addition of DNA ligase

Introductionof recombinant plasmid

into bacterial cells

Productionof multiple gene copies by

gene cloning
 Recombinant DNA
Technology
Summary of Recombinant DNA
Summary of Recombinant DNA
and Cloning (Fig.):
 Recombinant DNA
Technology

This
segment is "glued" into place
using an enzyme called DNA ligase.

Theresult is an edited, or
recombinant, DNA molecule.
 Recombinant DNA
Technology
When this recombinant plasmid DNA
is inserted into E. coli, the cell will be
able to process the instructions to
assemble the amino acids for insulin
production.
 Recombinant DNA
Technology

More importantly, the new

instructions are passed along to the
next generation of E. coli cells in the
process known as gene cloning.
Assignment: Human gene cloning pg
63
 Recombinant DNA
Technology

Fig:
Inserting
a DNA
sample
into a
Plasmid
 Recombinant DNA
Technology
References

http://en.wikipedia.org/wiki/Restriction_enzyme
http://web.mit.edu/esgbio/www/rdna/cloning.html
http://faculty.plattsburgh.edu/donald.slish/Transformation.ht
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