SPECIAL TECHNIQUES IN GENETICS

THAT CONTRIBUTE TO SOCIETY

GENE CLONING

GENE THERAPY
HUMAN GENOME PROJECT
PCR
RFLP BIO 511
 LESSON LEARNING OUTCOME
1. Define and Differentiate the concept of
Gene Cloning and Animal Cloning
2. State & Explain the application of Gene
Therapy in treating inherited genetic disorder
3. State the goal and benefits of HGP
4. Explain and characterize the techniques of
PCR & RFLP and its contribution towards
society

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 GENE CLONING: DEFINITION
Gene cloning is a process by which large
quantities of a specific, desired gene or
section of DNA may be cloned or copied once
the desired DNA has been isolated.

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GENE CLONING PROCEDURE

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 Method of Gene Cloning
1. The gene or DNA that is desired is isolated using restriction enzymes.
2. Both the desired gene and a plasmid are treated with the same restriction
enzyme to produce identical sticky ends.
3. The DNAs from both sources are mixed together and treated with the enzyme
DNA ligase to splice them together.
4. Recombinant DNA, with the plasmid containing the added DNA or gene has been
formed.
5. The recombinant plasmids are added to a culture of bacterial cells. Under the
right conditions, some of the bacteria will take in the plasmid from the solution
during a process known as transformation.
6. As the bacterial cells reproduces (by mitosis), the recombinant plasmid is copied.
Soon, there will be millions of bacteria containing the recombinant plasmid with its
introduced gene.
7. The introduced gene can begin producing its protein via transcription and
translation.
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GENE CLONING: PROS AND CONS
• The cloning of plants has many
PLANTS applications, not only to agriculture but to
medicine as well.
• Herbicide resistant crops, earlier/delayed
fruit ripening, longer lasting fruit and
higher yields of crops can be and have
been achieved through cloning.
• However, there are always disadvantages.
Mainly the cost and time needed for the
process or consumer resistance.
• Another disadvantage is that the
genetically altered offspring might not be
able to reproduce naturally.

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GENE CLONING: PROS AND CONS
The cloning of animals could/can:
ANIMALS 1. improve transgenic animals for
biotechnology
2. reduce numbers of animals necessary for
medical studies.
3. It will be possible to create large numbers of
custom-designed transgenic animals that
secrete medically useful human proteins in
their milk eg. human clotting factor and
fibrinogen.
4. Cloning can be used to create model animals
for research
eg. sheep designed to replicate the effects of
human cystic fibrosis

• However, animal rights, health and safety

concerns, cost and technical barriers are all
aspects that need to be taken into account. 9
GENE CLONING: PROS AND CONS
1. Cloning might reveal how the environment within the cells
HUMAN of the early embryo regulates gene function.
2. Might help combat genetic diseaes by allowing researches
to turn off bad genes and turn on good genes.
3. Grow organs intended for transplant therapy.
4. Aid infertile people
5. Can assess how much environment and genetic makeup
interact
eg. How much does environment play in governing
intelligence.
1. Alter functions of proteins eg. enzymes can be developed
on a large scale with new or improved functions.

• However, many people believe that cloning might

change the way humans reproduce, destroy human
dignity. Others believe it is unethical and a violation of
human rights. There is also the cost and technical
barriers. 10
 Gene Cloning: Step 1
• In order to clone a gene the first step is to isolate it using restriction
enzymes. These enzymes recognize specific regions on the DNA
molecule. The region of DNA shown below is from Rhodobacter
sphaeroides.
• The gene of interest lies in the region of the chromosome indicated in
blue. The base sequences shown are the ones that the restriciton
enzyme EcoRI recognizes.
• Note that reading from left to right in the top strand (GAATTC) is the
same as reading from right to left in the bottom strand (CTTAAG)
• Use EcoRI (RE) to cut the sugar-phosphate backbone at the points
indicated by the red arrows.

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 Gene Cloning: Step 2
• Unpaired bases result when EcoRI cuts a DNA molecule. Note that the
gene of interest is bounded by fragments of DNA containing unpaired
bases or "sticky ends".

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• If the temperature is lowered and DNA ligase is added, these unpaired
bases can reanneal following the rules of base pairing.

Temperature lowered
+ DNA Ligase

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 Gene Cloning: Step 3
• Plasmids can also be cut by restriction
enzymes if they have sites that the
restriction enzymes can recognize.

• For example: The plasmid pK19,

carries two useful genes,

1. Kan, conferring resistance to the pK19

antibiotic kanamaycin
2. lacZ, encoding the enzyme beta-
galactosidase which catalyzes the
hydrolysis of sugar.
3. The plasmid has a single recognition
site, within the lacZ gene, for the
restriction enzyme used.
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 Gene Cloning: Step 4

• When pK19 is cut by EcoRI it has

"sticky ends" that are
complementary to those made
by cutting R. sphaeroides.

• Like R. sphaeroides the "sticky

ends" can reanneal if DNA ligase
is added. This would return the pK19
plasmid to it's original ring
structure.

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 Gene Cloning: Step 5
• Note the complementarity
of the "sticky ends" of the
pK19 and the R.
sphaeroides DNA.
• Cool, add DNA ligase and
the molecules can
reanneal. Resulting in a
variety of recombinant
forms.
• The one of interest is the
plasmid containing the R. pK19
sphaeroides DNA.

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 Gene Cloning: Step 6

• This recombinant plasmid pKC105

contains both plasmid DNA and DNA
from R. sphaeroides.

• What are the difference between pK19

and pKC105?
pKC105
• Once you have created the plasmid it
is necessary to determine if you have
plasmids with the insert, reannealed
plasmids (like the original), or other
unexpected plasmids.
Step 7 
Transformation of bacteria using the
mixture of plasmids.
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 ANSWER

pKC105 pK19

1. Note that the recombinant plasmid pKC105 has two EcoRI sites. The host
plasmid pK19 only has a single EcoR1 site.

2. Inserting the R. sphaeroides DNA disrupts the base pair sequence in the
region of the plasmid chromosome that codes for the alpha peptide.
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 Gene Cloning: Step 7
(TRANSFORMING BACTERIA)
• ELECTROPORATION can be used to insert the recombinant
plasmid which also contained a gene for kanamycin resistance
(antibiotic resistance) into E. coli
• TRANSFORMATION  The process where the bacterial cells take
up the recombinant plasmids.
• The EcoRI site lies in the middle of the gene lacZ which encodes
beta galactosidase. Plasmids containing R. sphaeroides DNA will
NOT be able to produce beta galactosidase. Therefore, the
TRANSFORMED bacteria are also unable to make the alpha
peptide.
• These bacteria are called [lacZ-]  UNABLE TO HYDROLYZE
LACTOSE.

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 Gene Cloning: Step 8
(DETERMINE THE BACTERIA THAT CARRY PLASMID
CONTAINING R. sphaeroides DNA )
• We can plate out the transformed bacteria on a solid nutrient medium
containing kanamycin and a sugar called X-gal.

• Only bacteria that have the kanamycin-resistance plasmid will grow.

• The X-gal in the medium is used to identify plasmids that carry
R. sphaeroides DNA.

Bacteria with plasmids containing R.

sphaeroides foreign DNA are white
because they lack beta-galactosidase.

Bacteria with plasmids lacking R.

sphaeroides DNA stain blue when
beta-galactosidase hydrolyzes X-gal.

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 (1) CLONING ANIMAL (SCNT)
1. SOMATIC CELL NUCLEAR
TRANSFER
• Dolly was created using the technique
of somatic cell nuclear transfer
(SCNT)
• To do this, cells are taken from the
animal that is going to be cloned. In
the case of Dolly the sheep, a cell was
taken from her udder. These are
normal body cells - somatic cells - and
the nucleus is removed.
• The nucleus contains all of the genetic
material to make the animal and so, is
termed the donor cell.
• Egg cells are used for cloning because
of their ability to grow rapidly. The
egg cell’s nucleus is removed and the
nucleus from the donor cell is
inserted in its place.
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 (1) CLONING ANIMAL (SCNT)
• The egg is then stimulated to grow by
numerous stimulants which activates
the reconstructed embryo to divide
and grow. The division of the egg cell
follows the same process that would
occur if the egg was fertilised by sperm
during natural reproduction.
• The cell division continues for 5 days
until a blastomere forms. A blastomere
is a ball of nearly 100 cells all with the
same genetic material as the donor.
• Then it was implanted in the uterus of
surrogate mother.
• When Dolly was born, she was the only
lamb born from 277 attempts.

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Somatic Cell Nuclear Transfer (SCNT)

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 (2) CLONING ANIMAL (ES)

2. EMBRYO SPLITTING
• Another cloning technique is embryo splitting.
• Using microsurgery (surgery conducted under a microscope), an
embryo is split while it still consists of only a few cells.
• Genetically identical individuals develop from each portion in the
same way as identical twins are formed in nature.
• This technique has been used to successfully create cloned embryos
and cloned animals.

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(2) CLONING ANIMAL (ES)

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(2) CLONING ANIMAL (ES)

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CLONING ANIMAL (ES)
Researchers used a technique called embryo splitting
to produce this rhesus monkey, a female named Tetra,
at the Oregon Regional Primate Research Center, USA.

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 (3) CLONING ANIMAL (CT)
3. CHROMATIN TRANSFER

• There are a number of problems associated with nuclear transfer -

the method used to clone Dolly and almost all cloned animals since
then.

• When the nucleus is transferred to a new egg cell, the egg

reprograms the incoming nucleus to allow it to go back to its
undifferentiated state.
• Because it has come from an adult cell, it no longer needs to
produce the proteins, hormones and other molecules associated
with it being an embryo and growing to produce all the different
tissues in a whole body.

• Chromatin transfer is a new cloning technique aimed at reducing

these problems. It involves treating the cell of the animal to be
cloned to remove molecules associated with cell differentiation
before the nucleus is removed.
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CLONING ANIMAL (CT)
Tabouli and Baba Ganoush were the first cat clones produced using
chromatin transfer born in June 2004. They are clones of Tahini, a one
year old female Bengal cat.

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 GENE THERAPY
Methods for transferring a specific genes into mammalian cells
WHAT? to treat genetic disorders

• Researchers may use one of several approaches for correcting

HOW? faulty genes:
 A normal gene may be inserted into a nonspecific location
within the genome to replace a non-functional gene. This
approach is most common.
 An abnormal gene could be swapped for a normal gene through
homologous recombination.
 The abnormal gene could be repaired through selective reverse
mutation, which returns the gene to its normal function.
 The regulation (the degree to which a gene is turned on or off)
of a particular gene could be altered.

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HOW DOES GENE THERAPY WORKS?
• In most gene therapy studies, a "normal"
gene is inserted into the genome to replace
an "abnormal," disease-causing gene.
• A carrier molecule called a vector must be
used to deliver the therapeutic gene to the
patient's target cells. Currently, the most
common vector is a virus that has been
genetically altered to carry normal human
DNA.
• Target cells such as the patient's liver or
lung cells are infected with the viral vector.
• The vector then unloads its genetic material
containing the therapeutic human gene into
the target cell.
• The generation of a functional protein
product from the therapeutic gene restores
the target cell to a normal state.
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 THE FIRST
GENE THERAPY
• Example: Gene therapy for Severe
Combined Immunodeficiency (SCID)
• The first gene therapy trials began in
1990, treatment of a young girl,
Ashanti De Silva who has genetic
disorders (SCID)

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 Severe Combined
Immunodeficiency (SCID)
• The defining feature of SCID, commonly known as "bubble boy" disease,
is a defect in the specialized white blood cells (B- and T-lymphocytes)
that defend us from infection by viruses, bacteria and fungi.
• It is caused by a mutation in the gene encoding the enzyme adenosine
deaminase (ADA gene)

David Vetter died in 1984

at the age of 12 after a
failed operation that forced
him out of his bubble. Now,
Vetter’s disease is treatable
90 percent of the time. 33
 SCID
TREATMENT BY
GENE THERAPY

• Treatment started with isolation of white blood cells called T cells from the
patient
• T cell (part of the immune system) are mixed with a genetically modified
retrovirus carrying a normal copy the ADA gene
• The virus infects the T cell and inserts a normal copy of the ADA gene into
the cells’ genome
• The genetically modified T cells are grown in the laboratory in order to
ensure the gene is active
• The patient is treated by injecting the altered T cells into the bloodstream
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SCID TREATMENT BY GENE THERAPY

Adenovirus is often used by researchers to deliver the correct

gene to cells. Viruses deposit their own genetic material into
host cells to instruct those cells to make more viruses. In gene
therapy, the DNA for the desired gene is inserted into the
genetic material of the virus. The virus is engineered so
that it cannot reproduce, but it does deliver its new genetic
material which contains the desired DNA. 35
PROBLEMS & FAILURES IN GENE THERAPY

1. Integration of the retroviral genome into the

host cell’s genome occurs only if the host cell
are replicating their DNA
2. Insertion of viral genomes into the host
chromosomes can inactivate or mutate an
indispensable gene
3. Retroviruses having low cloning capacity. It
cannot carry sequences larger than 8 kb

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 HUMAN GENOME PROJECT
• The Human Genome Project was an international research
effort to determine the sequence of the human genome and
identify the genes that it contains.

• The Project was coordinated by the National Institutes of Health and

the U.S. Department of Energy. Additional contributors included
universities across the United States and international partners in the
United Kingdom, France, Germany, Japan, and China.
• The Human Genome Project formally began in 1990 and was completed
in 2003, 2 years ahead of its original schedule.
• A genome is an organism’s complete set of DNA, including all of its
genes.
• Each genome contains all of the information needed to build and
maintain that organism.
• In humans, a copy of the entire genome—more than 3 billion DNA base
pairs—is contained in all cells that have a nucleus.
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 Goals of the Human Genome Project

• Project goals were to:

1. Identify all the approximately 20,000-25,000 genes in human
DNA,
2. Determine the sequences of the 3 billion chemical base pairs
that make up human DNA,
3. Store this information in databases,
4. Improve tools for data analysis,
5. Transfer related technologies to the private sector, and
6. Address the ethical, legal, and social issues (ELSI) that may
arise from the project

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 ACCOMPLISHMENTS

• Provide a wealth of information that aid in the understanding of basic

biological processes.

• Scientists are now more effectively able to study gene

function and explore new areas of research such as how
human variation contributes to different diseases
worldwide.

• We have learnt that the human genome is made of 3.2 billion nucleotide
bases (of which there are four types: A, C, T, G). It is thought that over
30,000 genes are encoded by this sequence. 41
 » We have also discovered that over 50% of
the human genome is repetitive sequence
that does not code for any proteins and
the function of this large portion of “junk”
DNA is still puzzling scientists.

• Genome sequence information has helped scientists more

easily identify candidate disease genes, however, we also
realize that over 50% of the genes discovered in the human
genome are still classified as having unknown function.

• Human genome sequence information reveals that genome

sequences from person to person are almost (99.9%)
identical.

• Interestingly, comparative genomics shows 95% sequence

similarity between the human and chimpanzee genomes.
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 Brooker, Chapter 18, pg. 495

POLYMERASE CHAIN REACTION (PCR)

(PCR
• Polymerase chain reaction (PCR) is a molecular biological
WHAT? technique for amplifying (creating multiple copies of) DNA
without using a living organism, such as E. coli or yeast.

• The technique allows a small sample of DNA to be copied

multiple times so it can be used for analysis.

• PCR technique was developed in 1986 by Kary Mullis

• PCR is commonly used in medical and biological

APPLICATION research labs for a variety of tasks, such as the
detection of hereditary diseases, the identification of
genetic fingerprints, the diagnosis of infectious
diseases, the cloning of genes, and paternity testing.

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POLYMERASE CHAIN REACTION (PCR)
(PCR
• PCR is used to amplify a short, well-
defined part of a DNA strand. This can be
a single gene, or just a part of a gene.
• PCR, as currently practiced, requires 5
major components:
 DNA template, which contains the
region of the DNA fragment to be
amplified
 Two primers, which determine the
beginning and end of the region to be
amplified
 Taq DNA-Polymerase, which copies the
region to be amplified
 Nucleotides, from which the DNA-
Polymerase builds the new DNA
 Buffer, which provides a suitable
chemical environment for the DNA-
Polymerase 44
POLYMERASE CHAIN REACTION (PCR)
(PCR PROCESS
1. DNA to be amplified is denatured into
single strands. Heating at 90-950C
denatures the double-stranded DNA
which dissociates into single strands.
Usually takes 5 minutes
2. The temperature of the reaction is
lowered between 500C -700C. At this
temperature, the primers bind to the
denatured DNA. The annealing primers
serve as starting points for synthesizing
new DNA strands complementary to
the target DNA
3. Taq polymerase is added to the
reaction mixture. DNA synthesize is
carried out at temperatures between
700C and 750C.
4. The Taq polymerase extends the
primers by adding nucleotides in the 5’
to 3’ direction, making a double
stranded copy of the target DNA
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POLYMERASE CHAIN REACTION (PCR)
(PCR PROCESS

Schematic animation of the PCR cycle. (1) Denaturing at 95°C. (2) Annealing at of primers.
(3) Elongation by Taq Polymerase (4) The first cycle is complete. The two resulting DNA
strands make up the template DNA for the next cycle, thus doubling the amount of DNA
duplicated for each new cycle.
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RESTRICTION LENGTH POLYMORPHISM (RFLP)
• Technique in which organisms may be differentiated by
WHAT? analysis of patterns derived from cleavage of their DNA

RFLP analysis indirectly detects certain differences in

DNA nucleotide sequences HOW ?
1. After treating long DNA molecules with a restriction enzyme, the fragments
can be separated by size via gel electrophoresis.
2. This produces a series of bands that are characteristic of the starting molecule
and that restriction enzyme.
3. The separated fragments can be recovered undamaged from gels, providing pure
samples of individual fragments.

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RFLP ANALYSIS ON TWO
DIFFERENT ALLELES

We can use restriction fragment analysis to compare two different DNA molecules
representing, for example, different alleles.
•Because the two alleles must differ slightly in DNA sequence, they may differ in one or
more restriction sites
•If they do differ in restriction sites, each will produce different-sized fragments when
digested by the same restriction enzyme.
•In gel electrophoresis, the restriction fragments from the two alleles will produce different
band patterns, allowing us to distinguish the two alleles. Restriction fragment analysis is
sensitive enough to distinguish between two alleles of a gene that differ by only base pair
in a restriction site. 49
RESTRICTION LENGTH POLYMORPHISM (RFLP)
 Gel electrophoresis combined with nucleic acid hybridization allows
analyses to be conducted on the whole genome, not just cloned and
purified genes
 Although electrophoresis will yield too many bands to distinguish
individually, we can use nucleic acid hybridization with a specific probe
to label discrete bands that derive from our gene of interest.
 When probes encounter a complementary sequence of nucleotides in a
test sample of DNA, they bind to it by Watson-Crick base pairing and thus
identify it!
 The radioactive label on the single-stranded probe can be detected by
autoradiography, identifying the fragments that we are interested in.

 Probe is a molecule of single-stranded that is :

WHAT is Complementary to a run of nucleotides in one or more of
PROBE? the restriction fragments and is radioactive (fluorescent).

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 Gel electrophoresis
combined with nucleic
acid hybridization
allows analyses to be
conducted on the whole
genome, not just
cloned and purified
genes

We can tie together several molecular techniques to compare DNA samples from three
individuals.
1.We start by adding the restriction enzyme to each of the three samples to produce
restriction fragments.
2.We then separate the fragments by gel electrophoresis.
3.Southern blotting (Southern hybridization) allows us to transfer the DNA fragments
from the gel to a sheet of nitrocellulose paper, still separated by size.
4.This also denatures the DNA fragments.
5.Bathing this sheet in a solution containing our probe allows the probe to attach by base-
pairing (hybridize) to the DNA sequence of interest and we can visualize bands containing
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the label with autoradiography.
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RFLP in DNA FORENSIC
1. The image (courtesy of Lifecodes Corporation)
shows the test results in a rape case. Two
probes were used: one revealing the bands at
the top, the other those at the bottom.
2. DNA was tested from semen removed from
the vagina of the rape victim (EVIDENCE #2);
3. A semen stain left on the victim's clothing
(EVIDENCE #1);
4. the DNA of the victim herself (VICTIM) to be
sure that the DNA didn't come from her cells;
5. DNA from two suspects (SUSPECT #1, SUSPECT
#2);
6. a set of DNA fragments of known and
decreasing length (MARKER). They provide a
built-in ruler for measuring the exact distance
that each fragment travels.
7. the cells of a previously-tested person to be
sure the probes are performing properly
(CONTROL).
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 LESSON LEARNING OUTCOME
1. Define and Differentiate the concept of
Gene Cloning and Animal Cloning
2. State & Explain the application of Gene
Therapy in treating inherited genetic disorder
3. State the goal and benefits of HGP
4. Explain and characterize the techniques of
PCR & RFLP and its contribution towards
society

54