Molecular

Cloning
Yuwono
Department of Microbiology FAMUS
judeyon@yahoo.com
 Learning Objectives
Menjelaskan prinsip kloning
molekul
Menjelaskan kegunaan kloning
molekul dalam bidang kedokteran
Menjelaskan DNA interest, vector,
enzim restriksi dan sel kompeten
The major difference between the chemical structures
of DNA and RNA is that they have different

(A) phosphate groups (B) sugar rings (C) length (D) phosphoester bond
 True or false

The G/C and A/T base parings are important for

1) holding the two DNA strands together _____
2) replicating DNA _____
3) the temperatures required for denaturing and
renaturing of two DNA strands. _______
4) Keeping the DNA helix with the same width _____
5) the length of the DNA molecule ______
6) Whether a DNA should be in A form or B form _____
 How do we know which DNA region is
important for a particular biological function?

For example, which genes decides the eye color?

Which gene decides the height of a person?

Which genes decide the sexual identity?

Whether a gene is mutated in a cancer patient?

Identify, separate, and manipulate a

specific DNA fragment
 Molecular Cloning

The process of inserting a piece of

DNA molecule of interest into a DNA
carrier (vector) in order to make
multiple copies of the DNA of
interest in a host cell such as
bacteria
Purposes of molecular cloning

Separate a gene from the other genes

Amplification of modified forms of genetic materials

Manipulation of a piece of DNA for further experiments

 Vector (DNA carrier)
Plasmids

Cosmids

YAC

Bateriophage

Virus
Enzymes that can cut DNA

Enzymes that can join DNA

 Restriction Endonucleases
The Molecular Scissors
Host enzymes that prevent the invasion of foreign
DNAs such as viral DNA, by cutting them up.
Restriction
These enzymes cut within the foreign DNAs, rather
than chewing them away from the ends.
Endonucleases
These enzymes recognize a specific DNA sequence
(4-12bp) which is twofold symmetry and cut both DNA
strands
GAATTC
Some enzymes make staggered cuts CTTAAG

CCCGGG
Some make even cuts
GGGCCC
S -- deoxyribose P -- phosphate groups

3 5

5 3
 Sticky end

Sticky end
The first cloning experiment done by Boyer and Cohen
 Vectors -- the DNA carriers
Capable of replicating in bacteria -- an origin of replication
Allow the vector as well as the foreign DNA to amplify in the host cell
1) Plasmids 2) Phages
Originof replication
Antibiotic-resistant genes
Allow the host to grow on
selective media: Can selectively
amplify this specific vector
in the host cell
Multiple cloning sites
Allow insertion of foreign DNA
 Vectors -- the DNA carriers
Plasmid as a vector

Host: E. coli

Vector size: usually about 3kb

Insert size: up to 20kb. usually below 5 kb

Insert select: functional inactivation of the

ability to resist an antibiotic
 DNA ligase covalently links two DNA strands

5 3
Restriction
enzyme

Ligase

3 5

Restriction
enzyme

Ligase
 It is really a pain to do replica plating!

DNA ligase
 Multiple cloning sites
-peptide or
N-terminus of
-galactosiase
 peptidewhencombinedwiththecarboxylterminus
ofgalactosiasecanmakeafunctionalgalactosidase

+++ galactosidase
_ peptide

_ Carboxyl
Terminus

peptide
+++ +
Carboxyl
Terminus
peptidewhencombinedwiththecarboxylterminus
ofgalactosiasecanmakeafunctionalgalactosiase

peptide Cterminusofgalactosiase

Xgal

Xgal
 +

peptide Cterminusofgalactosiase

Xgal

Xgal
 Phages as DNA carriers
Bacteriophages are natural vectors that transduce DNA
from one bacterial cell to another

A bacteriophage can be regarded as a virus for a

bacterial cell

A bacteriophage cannot live or reproduce without

getting inside a bacterial cell
 ,whichthen

burstoutofthebacteriumandkillit.
A single phage can infect and clear out many bacterial
cells and creat a plaque on a bacterial lawn

A plaque contains a homogeneous population of a

phage
 What is Cloning?

Cloning is the production of one

or more individual plants or
animals (whole or in part) that are
genetically identical to an original
plant or animal.
 How is therapeutic
cloning done?
First cloning: Dolly the Sheep showed up
on the scene in 1997. Cloning
technologies have been around for much
longer than Dolly, though.

How does one go about making an exact

genetic copy of an organism? There are a
couple of ways to do this: artificial
embryo twinning and somatic cell nuclear
transfer. How do these processes differ?
 1. Artificial Embryo
Twinning
Artificial embryo twinning: low-tech version of
cloning this technology mimics the natural
process of creating identical twins.
In nature, twins occur just after fertilization of
an egg cell by a sperm cell.
In rare cases: a zygote, tries to divide into a two-
celled embryo the two cells separate Each
cell continues dividing on its own, developing
into a separate individual within the mother.
Since the two cells came from the same zygote,
the resulting individuals are genetically
identical.
 1. Artificial Embryo
Twinning
Artificial embryo twinning uses the same
approach, but it occurs in a Petri dish instead
of in the mother's body.
This is accomplished by manually separating a
very early embryo into individual cells, and
then allowing each cell to divide and develop
on its own.
The resulting embryos are placed into a
surrogate mother, where they are carried to
term and delivered. Again, since all the
embryos came from the same zygote, they are
genetically identical.
 2. Somatic Cell Nuclear
Transfer (SCNT)
Uses a different approach than
artificial embryo twinning, but it
produces the same result: an exact
clone, or genetic copy, of an
individual. This was the method
used to create Dolly the Sheep.
What does SCNT mean? Let's take
it apart:
 2. Somatic Cell Nuclear
Transfer (SCNT)
Somatic cell: A somatic cell is
any cell in the body other than the
two types of reproductive cells,
sperm and egg. These are also
called germ cells. In mammals,
every somatic cell has two
complete sets of chromosomes,
whereas the germ cells only have
one complete set.
 2. Somatic Cell Nuclear
Transfer (SCNT)
Nuclear: The nucleus is like the
cell's brain. It's an enclosed
compartment that contains all the
information that cells need to form
an organism. This information
comes in the form of DNA. It's the
differences in our DNA that make
each of us unique.
 2. Somatic Cell Nuclear
Transfer (SCNT)
Transfer: Moving an object from one
place to another.
To make Dolly, researchers isolated a
somatic cell from an adult female sheep.
Next, they transferred the nucleus from
that cell to an egg cell from which the
nucleus had been removed. After a couple
of chemical tweaks, the egg cell, with its
new nucleus, was behaving just like a
freshly fertilized zygote. It developed into
an embryo, which was implanted into a
surrogate mother and carried to term.
 2. Somatic Cell Nuclear
Transfer (SCNT)
The lamb, Dolly, was an exact
genetic replica of the adult female
sheep that donated the somatic
cell nucleus to the egg.
She was the first-ever mammal to
be cloned from an adult somatic
cell.
 Is cloning an organism
the same as cloning a
gene?
Cloning animals - sheep, mice,
even house pets in the news.

Researchers want to cloning, or

identifying, genes that are
responsible for various medical
conditions or traits.
Whwt is the different?
 What is the difference?
Cloning an animal, or any other
organism, refers to making an exact
genetic copy of that organism.
Cloning a gene means isolating an
exact copy of a single gene from the
entire genome of an organism. Usually
this involves copying the DNA sequence
of that gene into a smaller, more
accessible piece of DNA, such as a
plasmid. This makes it easier to study
the function of the individual gene in
the laboratory
 Why Clone?

Research advances over the past decade

have told us that, with a little work, we
humans can clone just about anything we
want, from frogs to monkeys and probably
even ourselves!
So, we can clone things, but why would
we want to? Let's look at some of the
reasons people give to justify cloning.
Some of the reasons people
give to justify cloning.
Cloning for medical purposes
Reviving Endangered or
Extinct
Species
Reproducing a Deceased Pet
Cloning Humans?
 Why would anyone want to
clone humans?
Some reasons include:
To help infertile couples have
children
To replace a deceased child
 What Are Some Issues In
Cloning?
We saw that the success rate in cloning is quite low.
Even if we can increase the odds of success, problems can
arise during the clone's development, both before and
after pregnancy.
Despite these risks, supporters of human reproductive
cloning see it as a possible solution to infertility problems.
Others support therapeutic cloning to create embryonic
stem cells for research and medicine.
What are the possible implications of cloning to society?
All of us - researchers, policymakers and the public - have
a responsibility to explore the potential effects of cloning
technologies on our lives so that we can make informed
decisions.
 For each new application of
cloning technologies, we must
consider:
What are the benefits?
What are the risks?
Whom will the technology help?
Does it have the potential to hurt anyone?
What does this mean for me?
For my family?
For others around me?
Why might others not share my view?
 Ethical, legal and social
issues.
There are several types of issues to
consider as we think about cloning.
Ethical issues are those that ask us to
consider the potential moral outcomes of
cloning technologies.
Legal issues require researchers and the
public to help policymakers decide
whether and how cloning technologies
should be regulated by the government.
Social issues involve the impact of cloning
technologies on society as a whole
 What are the Risks of
Cloning?
When we hear of cloning successes, we learn
about only the few attempts that worked.
What we don't see are the many, many cloning
experiments that failed! And even in the
successful clones, problems tend to arise later,
during the animal's development to adulthood.
Cloning animals shows us what might happen if
we try to clone humans. What have these animals
taught us about the risks of cloning?
 What are the Risks of
Cloning?
High failure rate
Problems during later
development
Abnormal gene expression
patterns
Telomeric differences
 High failure rate
Cloning animals through somatic
cell nuclear transfer is simply
inefficient. The success rate
ranges from 0.1 percent to 3
percent, which means that for
every 1000 tries, only one to 30
clones are made. Or you can look
at it as 970 to 999 failures in 1000
tries.
 High failure rate
Why is this? Here are some reasons:
The enucleated egg and the transferred
nucleus may not be compatible
An egg with a newly transferred
nucleus may not begin to divide or
develop properly
Implantation of the embryo into the
surrogate mother might fail
The pregnancy itself might fail
 Problems during later
development
Cloned animals that do survive tend to be
much bigger at birth than their natural
counterparts. Scientists call this "Large
Offspring Syndrome" (LOS). Clones with LOS
have abnormally large organs. This can lead
to breathing, blood flow and other problems.
Because LOS doesn't always occur, scientists
cannot reliably predict whether it will
happen in any given clone. Also, some clones
without LOS have developed kidney or brain
malformations and impaired immune systems,
which can cause problems later in life.
Abnormal gene expression
patterns
In cloning, the transferred nucleus
doesn't have the same program as a
natural embryo.
It is up to the scientist to reprogram
the nucleus, like teaching an old dog
new tricks.
Complete reprogramming is needed for
normal or near-normal development.
Incomplete programming will cause the
embryo to develop abnormally or fail.
Abnormal gene expression
patterns
Are the surviving clones really clones? The clones look
like the originals, and their DNA sequences are
identical. But will the clone express the right genes at
the right time?
In Click and Clone, we saw that one challenge is to re-
program the transferred nucleus to behave as though it
belongs in a very early embryonic cell. This mimics
natural development, which starts when a sperm
fertilizes an egg.
In a naturally-created embryo, the DNA is programmed
to express a certain set of genes. Later on, as the
embryonic cells begin to differentiate, the program
changes. For every
type of differentiated cell - skin, blood, bone or nerve,
for example - this program is different.
 Telomeric differences
As cells divide, their chromosomes get
shorter. This is because the DNA sequences at
both ends of a chromosome, called telomeres,
shrink in length every time the DNA is copied.
The older the animal is, the shorter its
telomeres will be, because the cells have
divided many, many times. This is a natural
part of aging.
So, what happens to the clone if its
transferred nucleus is already pretty old? Will
the shortened telomeres affect its
development or lifespan?
 Telomeric differences

When scientists looked at the telomere lengths of

cloned animals, they found no clear answers.
Chromosomes from cloned cattle or mice had
longer telomeres than normal. These cells
showed other signs of youth and seemed to have
an extended lifespan compared with cells from a
naturally conceived cow. On the other hand,
Dolly the sheep's chromosomes had shorter
telomere lengths than normal. This means that
Dolly's cells were aging faster than the cells from
a normal sheep.
To date, scientists aren't sure why cloned
animals show differences in telomere length
 Some Question!!
Who has the right to have children, no matter how they
are created? Who doesn't? Why?
Is human cloning "playing with nature?" If so, how does
that compare with other reproductive technologies such as in
vitro fertilization or hormone treatments?
Does cloning to create stem cells, also called therapeutic
cloning, justify destroying a human embryo? Why, or why
not?
If a clone originates from an existing person, who is the
parent?
What are some of the social challenges a cloned child
might face?
Do the benefits of human cloning outweigh the costs of
human dignity?