Download Gene Cloning Technology

Gene Cloning Technology
Also known as:
Genetic engineering or Genetic manipulation (GM) technology
– implies precision engineering being applied
to DNA molecules
Recombinant DNA technology
- implies that new combinations of DNA
molecules can be made
i.e. “recombinant” DNA molecules
Overview of Genetic Engineering procedure
1.  Making recombinant DNA molecules that can replicate in
bacterial cells
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Genetic engineering applications
A tool kit for recombinant DNA technology
1.  Tools to cut DNA molecules into large “gene-sized” fragments
2.  Vector DNA molecules that can replicate and into which
“foreign” DNA can be inserted
3.  Tools to join vector DNA and “foreign” DNA together
to create recombinant DNA molecules
4. Methods for introducing “recombinant” DNA molecules into
bacterial cells where they can replicate
5.  A method for identifying bacterial cells that have taken up the
recombinant DNA molecules = a method of selecting for
transformants
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1.  Tools for cutting DNA molecules into “gene-sized” fragments
  A typical chromosome might contain more than 5 million base pairs
  Molecules of this enormous size cannot easily be cloned and they
contain 1000’s of genes
  A single gene might typicallly contain a few thousand base pairs of
DNA sequence. Molecules of this size can be easily cloned
  For this reason, we need to be able to cut the very large
chromosome-sized DNA molecules into smaller gene-sized
fragments
  This can be done using a cutting tool called a
restriction enzyme
Restriction enzymes
  Restriction enzymes are made by bacteria to protect themselves
incoming virus DNA
  They act as molecular scissors to cut DNA into large fragments
  They have a unique property:
they cut DNA in a sequence-specific manner by
recognizing a specific sequence of bases
e.g. The restriction enzyme EcoRI will only cut a DNA molecule
if it contains the following sequence:
G-A-A-T-T-C
C-T-T-A-A-G
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Restriction enzyme Mva1 (grey) is shown wrapped around DNA
(multicolored) (Kaus-Drobek et al. 2007). Protein database ID: 2OAA.
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Restriction enzymes cut DNA infrequently
The frequency with which this particular sequence of 6 base pairs
will occur in a very large DNA molecule can be calculated on the
basis of:
the probability of occurrence of any specified base at each position:
G AAT T C
¼
¼
¼
¼ ¼ ¼ = 1/ 46 = 1 in 4,100 bases pairs
Because this enzymes cuts DNA only once in every 4,000 base pairs
(on average), the fragments are sufficiently large to contain an
entire gene
DNA fragments have single-stranded “sticky” ends after being
cut by a restriction enzyme. The sequence of bases at the ends
of the fragment are unique for each type of restriction enzyme
G-A-A-T-T-C
C-T-T-A-A-G
G
C-T-T-A-A
A-A-T-T-C
G
single-stranded 4 base “sticky” ends
The single-stranded ends can anneal or base pair with the same
type of end on a DNA molecule from any source e.g. animal
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The DNA polymerase used for PCR is Taq polymerase. Taq
polymerase is derived from a heat-stable bacterium. It works in a
similar manner as the polymerase found in human cells.
www.odec.ca/projects/2005/anna5m0/public_html/methods.htm
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2. DNA Cloning Vectors
  A cloning vector is a DNA molecule that can contains information
for replicating itself – a so-called “origin of replication”
  If a piece of foreign DNA is incorporated into the vector, then the
foreign DNA will be replicated along with the vector DNA
  The most commonly used type of cloning vector is a small (4000 bp)
circular DNA molecule called a plasmid
  Plasmids are found naturally in various types of bacteria
  Plasmids can replicate independently of the bacterial chromosome
because they have an origin of replication
A typical plasmid DNA might have the following features
lacZ gene
for “colony colour”
selection
Antibiotic
resistance gene
Ampr
Any bacterial cell that
takes up the plasmid
will be resistant to
ampicillin
EcoRI restriction site
The plasmid DNA circle
can be opened up at this
site and foreign DNA
can be inserted into the
plasmid
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3. Making recombinant DNA molecules
  A recombinant DNA molecule is made by joining together
(a) a piece of foreign DNA (e.g. human DNA) and
(b) vector DNA (e.g. a plasmid)
  The two DNAs can be joined together by an enzyme called
DNA ligase
4. Cloning the recombinant DNA molecules
  The recombinant DNA molecules will replicate if they are
taken up by a bacterial cell and the bacterial cell will be
transformed to antibiotic resistance
  Cells that have not taken up a recombinant DNA molecule
will be killed by the antibiotic
Cut plasmid DNA
Cut human DNA
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Transformation of
bacteria with
recombinant DNA
Selection of transformed
bacteria on agar plates
containing ampicillin
and X-Gal
X-Gal is a b-galactosidase
chromogenic substrate. In the
presence of b-galactosidase
X-Gal is hydrolysed and
forms a blue precipitate.
Cloned human DNA
click4biology.info/c4b/4/gene4.4.htm
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Factor IX : A human
clotting factor is produced
by genetically modified
sheep. The protein(factor
IX) is expressed in milk
from which it must be
isolated before use
byhaemophiliacs.
click4biology.info/c4b/4/gene4.4.htm
Factor IX protein purified from CHO cells (a mammalian cell
line) engineered to express human Factor IX using the types
of cloning technologies described in this lecture.
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‘In the 1980s, researchers used genetic engineering to manufacture a human insulin. In 1982, the Eli
Lilly Corporation produced a human insulin that became the first approved genetically engineered
pharmaceutical product’.
Human insulin is grown in the lab inside common bacteria. Escherichia coli is a widely used type of
bacterium, but yeast is also used.
cited www.madehow.com/Volume-7/Insulin.html
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fhs-bio-wiki.pbworks.com/w/page/22957102/Recombinant DNA (plasmids)
Genetic Manipulation and Agriculture
  Rice is the major food source for more than a billion people
The problem:
Rice has two major nutritional problems if it is the major (or only)
part of a person’s diet:
- it is a poor source of iron
- it contains no vitamin A or proVitamin A
A diet deficient in Vitamin A causes irreversible blindness in children
and weakens their immune systems
A diet deficient in iron leads to anaemia which affects growth potential
and weakens the immune system
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  The solution: The Golden Rice project
The project was led by a Swiss Government Research centre
not by a Biotechnology Company
Aim: to genetically engineer rice
(i) to produce proVitamin A
(ii) to accumulate iron from the soil more efficiently
1. Genetic engineering for proVitaminA production
  Inserted genes from daffodil and bacteria that enable rice to
make carotenoids = proVitamin A (yellow orange pigments)
human metabolism can convert carotenoids into Vitamin A
2. Genetic engineering for increased iron accumulation
  Introduced 3 genes - a gene for increased iron uptake
- a gene for iron storage
- a gene that increases iron bio-availability
Golden Rice has been
donated “royalty-free”
to the International Rice
Research Institute in the
Phillipines for use in
third world countries
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Crops as biotechnology factories: “BioPharming”
1. Plants can be genetically engineered to produce important
therapeutic proteins e.g. vaccines
and proteins like insulin
2. There are major advantages in using plants for this purpose:
- plants are a very inexpensive production system
- very easy to scale-up the process
- alternative production methods are very expensive
e.g. production in animal cell cultures or
bacterial cultures
3. The recovered protein is free from animal viruses and other
pathogens
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