Cloning

Mr. Glen Mangali

DNA evidence was used to solve a double
murder in England
Showed that two murders could have been
committed by the same person
Showed the innocence of someone who confessed
to one of the murders
Showed the absence of a match in 5,000 men tested
when the murderer persuaded another man to
donate blood in his name
Showed a match with the murderer and DNA found
with both victims
Introduction:
DNA and Crime Scene Investigations
Copyright 2009 Pearson Education, Inc.
Colin Pitchfork Colin Pitchfork
GENE CLONING
Copyright 2009 Pearson Education, Inc.
Genes can be cloned in recombinant
plasmids
Genetic engineering involves manipulating
genes for practical purposes
Gene cloning leads to the production of multiple
identical copies of a gene-carrying piece of DNA
Recombinant DNA is formed by joining DNA
sequences from two different sources
One source contains the gene that will be cloned
Another source is a gene carrier, called a vector
Plasmids (small, circular DNA molecules
independent of the bacterial chromosome) are often
used as vectors
Copyright 2009 Pearson Education, Inc.
Steps in cloning a gene
1. Plasmid DNA is isolated
2. DNA containing the gene of interest is isolated
3. Plasmid DNA is treated with restriction enzyme that
cuts in one place, opening the circle
4. DNA with the target gene is treated with the same
enzyme and many fragments are produced
5. Plasmid and target DNA are mixed and associate
with each other
Copyright 2009 Pearson Education, Inc.
Genes can be cloned in
recombinant plasmids
6. Recombinant DNA molecules are produced when
DNA ligase joins plasmid and target segments
together
7. The recombinant DNA is taken up by a bacterial cell
8. The bacterial cell reproduces to form a clone of
cells
Copyright 2009 Pearson Education, Inc.
Genes can be cloned in
recombinant plasmids
Animation: Cloning a Gene
E. coli bacterium
Plasmid
Bacterial
chromosome
Gene of interest
DNA
Cell with DNA
containing gene
of interest
Isolate
plasmid
Isolate
DNA
1
2
E. coli bacterium
Plasmid
Bacterial
chromosome
Gene of interest
DNA
Cell with DNA
containing gene
of interest
Gene
of interest
Isolate
plasmid
Isolate
DNA
Cut plasmid
with enzyme
Cut cells DNA
with same enzyme
1
2
3
4
E. coli bacterium
Plasmid
Bacterial
chromosome
Gene of interest
DNA
Cell with DNA
containing gene
of interest
Gene
of interest
Isolate
plasmid
Isolate
DNA
Cut plasmid
with enzyme
Cut cells DNA
with same enzyme
1
2
3
4
Combine targeted fragment
and plasmid DNA
5
E. coli bacterium
Plasmid
Bacterial
chromosome
Gene of interest
DNA
Cell with DNA
containing gene
of interest
Gene
of interest
Isolate
plasmid
Isolate
DNA
Cut plasmid
with enzyme
Cut cells DNA
with same enzyme
1
2
3
4
Recombinant
DNA
plasmid
Gene
of interest
Combine targeted fragment
and plasmid DNA
Add DNA ligase,
which closes
the circle with
covalent bonds
5
6
Recombinant
DNA
plasmid
Gene
of interest
Recombinant
bacterium
Put plasmid
into bacterium
by transformation
7
Examples of
gene use
Recombinant
DNA
plasmid
E. coli bacterium
Plasmid
Bacterial
chromosome
Gene of interest
DNA
Gene
of interest
Cell with DNA
containing gene
of interest
Recombinant
bacterium
Clone
of cells
Genes may be inserted
into other organisms
Genes or proteins
are isolated from the
cloned bacterium
Harvested
proteins
may be
used directly
Examples of
protein use
Gene
of interest
Isolate
plasmid
1
Isolate
DNA
2
Cut plasmid
with enzyme
3
Cut cells DNA
with same enzyme
4
Combine targeted fragment
and plasmid DNA
5
Add DNA ligase,
which closes
the circle with
covalent bonds
6
Put plasmid
into bacterium
by transformation
7
Allow bacterium
to reproduce
8
9
What is Animal
Cloning?
It refers to the creation of
cells or whole animals using
DNA from a single parent,
bypassing the normal
reproductive process.
Types of Cloning
Therapeutic
cloning
Reproductive
cloning
also called "embryo cloning," is
the production of embryos, cells,
specific body parts and organs for
research and medical purposes.
The goal of this process is to
harvest stem cells that can be
used to study human
development and to treat disease.
production of an animal that
has the same nuclear DNA
as the existing animal by
using the process called
"somatic cell nuclear transfer"
(SCNT).
FYI
On September 15,
2007, the Southeast
Asias first cloned
water buffalo using
somatic cell nuclear
transfer was created
in the Philippines.
Uses of Cloning
To achieve a gene knockout
For production of
recombinant protein in a
mammalian cell culture, and
in the related technique of
pharming
In gene therapy, in which
human cells are engineered
in order to treat a disease
Gene therapy
a technique for correcting
defective genes responsible
for disease development.
A normal gene may be
inserted into a
nonspecific location
within the genome to
replace a nonfunctional
gene.
The abnormal gene could be
repaired through selective
reverse mutation
The regulation of a particular
gene could be altered.
An abnormal gene could
be swapped for a normal
gene through homologous
recombination.
Animal Cloning
Procedures
An unfertilized egg
cell (germ line cell)
is taken from one
animal (surrogate
organism) and has
its nucleus
removed making it
an enucleated egg.
A body cell
(somatic cell) is
taken from
another animal
(donor organism)
and its nucleus is
also removed and
reprogram.
Fusion of
enucleated egg
with the
reprogram
nucleus of the
donor cell by
applying an electric
current or using
polyethylene glycol
(PEG).
The fused cell is
cultured and
allowed to
divide until it
reaches a
suitable stage in
its development.
It can be implanted
to the uterus of the
surrogate organism
and allowed to
develop or it can be
used in embryo
cloning, since
embryonic cells are
totipotent.
ADVANTAGES
of Animal Cloning
Husbandry-efficient production of
transgenic animals.
Pharmaceuticals- production of milk
proteins
Medicine-through
xenotransplantation and therapeutic
cloning
Genetics- greater understanding of
the development of embryo, heredity,
genetic structure and ageing.
Biodiversity conservation- by
repopulating endangered animals
Economy- efficiency in the production
of food-producing animals
Personal- replacing lost or deceased
family pets.
DISADVANTAGES
of Animal Cloning
Small chances of successful
attempts
Very expensive/costly.
Cloned animals are susceptible
to premature illness and death
Difficulty in reprogramming
the nucleus of the donor cell.
Health risks from mutation of
genes.
Abuse of the technology
Lets have a debate
Are you in favor of
cloning?
ETHICAL ISSUES
Abortion for embryo
cloning.
Health of cloned animals
Future effects
TBANK
Y00
Quiz ( True or False)
1. Animal cloning refers to the creation of
cells or whole animals using DNA from a
two parents, by passing the normal
reproductive process.
2. An unfertilized egg cell is known as germ
line cell.
3. Somatic cell is known as sex cell
Quiz ( True or False)
4. Fused cell is cultured and allowed to unite
until it reaches a suitable stage in its
development.
5. One of the objective of animal cloning is to
achieve a gene knockout.
6. In cloning, normal gene may be inserted
into a nonspecific location within the
genome to replace a nonfunctional gene.
Quiz ( True or False)
7. Surrogate organism donates cells to be
cultured.
8. Animal cloning helps in the production of
milk proteins.
9. Fusion of enucleated egg is done by
applying magnetic force.
10. Gene therapy is a technique for correcting
defective genes responsible for disease
development.
Answer Key
1. F
2. T
3. F
4. F
5. T
6. T
7. F
8. T
9. F
10. T