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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, geneticallyengineered crop plants and
transgenic mice have become possible
because of the powerful techniques
developed to manipulate nucleic acids and
proteins.
Recombinant DNA Technology

In the 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

Currently it 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 doublehelical 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

Sticky end 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

If another 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 produce
cohesive ends ( Fig. 3.1):
Recombinant DNA Technology
Creating recombinant DNA :

The first 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
technology process:

This portion of the DNA is then coupled to
a carrier molecule.

The hybrid DNA is introduced into a
chosen cell for reproduction and synthesis.
Recombinant DNA Technology
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 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

The gene 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)

The bacteria are then spread over a plate
that contains ampicillin.
Recombinant DNA Technology
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

As long 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.
Recombinant DNA Technology
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:
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
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Addition of DNA ligase

Introduction of recombinant plasmid into
bacterial cells

Production of multiple gene copies by
gene cloning
Recombinant DNA Technology
Summary of Recombinant DNA and
Cloning (Fig.):
Recombinant DNA Technology
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This segment is "glued" into place using
an enzyme called DNA ligase.

The result 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
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http://en.wikipedia.org/wiki/Restriction_enzyme
http://web.mit.edu/esgbio/www/rdna/cloning.html
http://faculty.plattsburgh.edu/donald.slish/Transformation.html