7 biotechnology and human disease

Biotechnology and
Associated
Human Diseases

Learning Objectives
Upon completion of this course, you will have
gained an understanding of techniques that are
currently being utilized in the biotechnology
and pharmaceutical industries.
You will be familiar with concepts pertaining to
basic molecular biology principles and
techniques for understanding various areas of
research and their applications.

Topic Outline
- X-ray Diffraction Reveals the Molecular Structure of
DNA
- Chromatography Electrophoresis
- Ultracentrifugation
- SOUTHERN BLOTTING
- POLYMERASE CHAIN
- ANALYSIS OF GENE EXPRESSION

ln the past, efforts to understand genes and their
expression have been confounded by the immense
size and complexity of human DNA.
• The human genome contains DNA with
approximately three billion (109) base pairs that
encode 30,000 to 40,000 genes located on 23
pairs of chromosomes.
• It is now possible to determine the nucleotide
sequence of long stretches of DNA, and
essentially the entire sequence of the human
genome is now known.
• This effort (called the Human Genome Project)
was made possible by several techniques that
have already contributed to our understanding of
many genetic diseases.

I- X-ray Diffraction
Reveals the Molecular
Structure of DNA

Photo 51 is the nickname given to an X-ray diffraction
image of DNA taken by Rosalind Franklin in 1952 that
was critical evidence in identifying the structure of
DNA.
• The photo was taken by Franklin while working at
King's College London in Sir John Randall 's group.
Crystallography is the experimental science of the
arrangement of atoms in solids.
• Crystallographic methods now depend on the analysis
of the diffraction patterns of a sample targeted by a
beam of some type.
• Crystallographers often explicitly state the type of
illumination used when referring to a method, as with
the terms X-ray diffraction, neutron diffraction and
electron diffraction.

The second piece of evidence available to Watson and Crick was
the X-ray diffraction pattern obtained when a crystallized DNA
fiber is bombarded with X-rays.
The theory behind X-ray diffraction analysis is very complex but
is based on the fact that:
the angles at which X-rays are deflected on passage through a
crystal will be determined by the three-dimensional structure
of the molecules in the crystal
after passage through the crystal, to expose a photographic film.
The result is a pattern of spots, the positions and intensities of
which may allow the structure of the molecule to be deduced.

Photo 51, an X-ray
diffraction image of
sodium salt of DNA.

Chromatography techniques are used to separate
compounds on the basis of their relative
affinities for absorption on to a solid matrix.
o They are particularly useful for separating
individual proteins, amino acids or nucleolides
In paper chromalography, this matrix is a strip of
filter paper A ,sample of the compounds to be
analysed is placed at one end of the paper strip
and eluted by soaking an aqueous or organic
solution (the solvent) along the strip,
As the solvent soaks along the paper it carries the
compounds with it, but at diffrent rates depending
on the relative affinities of the compounds for
absorption on to the matrix,

 Chargaffs method for analysing the base composition of
DNA involved :
o first breaking the molecule into its component
nucleotides by treatment with acid or alkali
o The sample was then eluted along a paper strip with
any of several solvents and
o the resulting spots cut out
o The pure nucleotides were recovered from the paper by
soaking in an aqueous solution, and their concentrations
measured.
 Chromatography is now more routinely carried out in a
glass column packed with tiny beads, composed of
substances such as cellulose or agarose, immersed in
the solvent
The sample is layered on to the top of the column and
eluted by passing through more solvent.
The separated compounds are then collected as they drip
out of the bottom of the column.

Prior to the fraction collection, the samples that
are eluted from the column pass through a
detector such as a spectrophotometer or mass
spectrometer so that the concentration of the
separated samples in the sample solution
mixture can be determined.

Column chromatography proceeds by a series of steps

An automated ion chromatography system

Typical set up for manual
column chromatograph

• In an aqueous solution the role of the migration of a molecule
depends on two factors:
its shape and its electrical charge,
when DNA molecules are placed in an eleclrical field they will
migrate towards the positive pole.
 Nowadays, Billion Crystallography X-ray diffraction,
neutron diffraction, electron diffraction, Chromatography
electrophoresis of DNA is usually carried out in a gel made of
agarose, polyacrylamide or a mixture of the two
 In a gel, the migration role of a macromolecule is influenced
by a third factor, its size
This is because the gel comprises a complex network of pores
through which the molecules must travel to reach the
electrode.
The smaller the faster it can migrate through the gel
Gel will therefore separate DNA molecules to size

The measurement and analysis are mostly
done with a specialized gel analysis
software.
Capillary electrophoresis results are typically
displayed in a trace view called an
electropherogram.

The ullracenlrifuge, Invented by Svedberg in
1925
Allows samples to be subjected to centrifugal
forces, up to several hundred thousand xg cell
components and macromolecules sediment
during ultracentnfugation
at a rate dependent on their
size, shape, density and molecular weight

Two versions of ultracentrifugation are now
important in studying DNA
velocity sedimentation analysis,
involves measuring the rate at which a
macromolecule or particle sediments through a
dense solution, and is expressed as a
sedimentation coefficient.
The rate of sedimentation is a measure of
the size of the molecule or particle
(although shape and density also
influence the rate).

Density gradient centrifugation
centrifuging a dense solution (usually of
caesium chloride (CsCI)), as a high centrifugal
force will pull the caesium and chloride ions
towards the bottom of the tube.
Macromolecules present in the CsCI solution
when it is will form bands at distinct points in
the gradient the exact position depending on
the buoyant macromolecule,
DNA has a buoyant density of about 1.7 g cm3
therefore migrate to the point CsCI density is
also 1.7 g cm3

In physics, buoyancy (pronounced /ˈbɔɪ.ənsi/) is
an upward acting force exerted by a fluid, that
opposes an object's weight.

Spectroscopy involves analysis of substances by the
spectra they produce,
Spectroscopy is the study of the interaction
between matter and radiated energy.
The spectrum of light emitted or absorbed by a
substance is characteristic of the substance,
o DNA strongly absorbs ultraviolet radial ion with a
wavelength of 260 nm,
o proteins on the other hand have a strong
absorbance at 280 nm
Ultraviolet spectroscopy is often used to check
that samples of DNA obtained from living cells
are pure and do not contain protein or other
contaminants and
to determine the concentration of DNA in a
sample.

Analysis of white light by
dispersing it with a prism is
example of spectroscopy

Biotechnology
It is now possible to determine the nucleotide
sequence of long stretches of DNA, and
essentially the entire sequence of the human
genome is now known.
This effort (called the Human Genome Project)
was made possible by several techniques that
have already contributed to our understanding
of many genetic diseases.

I. Restriction Endonucleases
Restriction enzymes are DNA-cutting enzymes
found in bacteria
(and harvested from them for use).
Restricion enzymes are used experimentally to
obtain precisely defined DNA segments called
restriction fragments.

"Sticky" and "blunt" ends
Restriction enzymes cleave DNA so as to produce a
3'-hydroxyl group on one end and a 5'-phosphate
group on the other.
Some restriction endonucleases, such as Taql,
form staggered cuts that produce "sticky" or
cohesive ends-that is, the resulting DNA
fragments have single-stranded sequences that are
complementary to each other.
 Other restriction endonucleases, such as Haelll,
cleave in the middle of their recognition sequence
that is, at the axis of and produce fragments that
have "blunt" ends that do not form hydrogen
bonds with each other.

Using the enzyme DNA ligase,
sticky ends of a DNA fragment of interest
can be covalently joined with other DNA
fragments that have sticky ends produced
by cleavage with the same restriction
endonuclease
The hybrid combination of two fragments
is called
a recombinant DNA molecule.

Restriction sites
Restriction site:
DNA sequence that is recognized by
a restriction enzyme.
Hundreds of these enzymes, having different
cleavage specificities (varying in both
nucleotide sequences and length of recognition
sites), are commercially available as analytic
reagents.

More commonly, to clone a nucleotide sequence of interest,
 the total cellular DNA is first cleaved with a specific
restriction enzyme, creating hundreds of thousands of
fragments. Therefore,
individual fragments cannot be isolated.
1. Instead, each of the resulting DNA fragments is joined to a
DNA vector molecule (cloning vector) to form a hybrid
molecule.
2. Each hybrid recombinant DNA molecule conveys its inserted
DNA fragment into a single host cell, for example, a
bacterium, where It is replicated (or "amplified" ).
3. As the host cell multiplies, it forms a clone in which every
bacterium carries copies of the same inserted DNA fragment,
hence, the name "cloning"
4. The cloned DNA is eventually released from its vector by
cleavage (using the appropriate restriction endonuclease)
and is isolated.

A.vector :
It is a molecule of DNA to which the fragment of DNA
to be cloned is joined.
- Essential properties of a vector Include:
1) It must be capable of autonomous replication within a
host cell,
2) it must contain at least one specific nucleotide
sequence recognized by a restriction endonuclease
3) It must carry at least one gene that confers the ability to
select for the vector, such as an antibiotic resistance
gene.
Commonly used vectors include plasmids and bacterial
and animal viruses.

B.DNA libraries
DNA library : Is a collection of cloned restriction
fragments of the DNA of an organism.
Two kinds of libraries will be discussed:
1. Genomic DNA libraries:
A genomic library is the collection of fragments of double-
stranded DNA obtained by digestion of the total DNA of
the organism with a restriction endonuclease and
subsequent ligation to an appropriate vector.
Genomic library : The amplified DNA fragments represent
the entire genome of the organism.

2. Complementary DNA (cDNA) libraries:
 If a gene of interest is expressed at a very high
level in a particular tissue,
 it is likely that the mRNA corresponding to
that gene is also present at high concentrations
in the cell.
This mRNA can be used as a template to make
a complementary double-stranded DNA
(cDNA) molecule using the enzyme reverse
transcriptase.
The resulting cDNA is thus a double-stranded
copy of mRNA.

C. Sequencing of cloned DNA fragments
The base sequence of DNA fragments that have
been cloned and purified can be determined in
the laboratory.
The original procedure for this purpose was the
Sanger dideoxy method

The dideoxy method gets its name
from the critical role played by
synthetic nucleotides that lack the
-OH at the 3′ carbon atom (red
arrow).
A dideoxynucleotide
(dideoxythymidine triphosphate —
ddTTP — is the one shown here)
can be added to the growing DNA
strand but when it is, chain
elongation stops because there is
no 3′ -OH for the next nucleotide to
be attached to.
For this reason, the dideoxy method
is also called the
chain termination method.

The Procedure
 The DNA to be sequenced is prepared as a single strand.
 This template DNA is supplied with a mixture of all four
normal (deoxy) nucleotides in ample quantities
– dATP
– dGTP
– dCTP
– dTTP
 a mixture of all four dideoxynucleotides, each present in
limiting quantities and each labeled with a "tag" that
fluoresces a different color:
– ddATP
– ddGTP
– ddCTP
– ddTTP
 DNA polymerase I

• Because all four normal nucleotides are
present, chain elongation proceeds normally
until, by chance, DNA polymerase inserts a
dideoxy nucleotide (shown as colored letters)
instead of the normal deoxynucleotide (shown
as vertical lines).
 At the end of the incubation period, the
fragments are separated by length from longest
to shortest.
 Each of the four dideoxynucleotides fluoresces
a different color when illuminated by a laser
beam and an automatic scanner provides a
printout of the sequence

How can a specific gene or DNA sequence of
interest be picked out of the mixture of thousands
or even millions of irrelevant DNA fragments?
The answer lies in the use of a probe-
a single-stranded piece of DNA, labeled with a
radioisotope, such as 32p, or
with a non-radioactive probe, such as biotin.
The nucleotide sequence of a probe is
complementary to the DNA of interest, called the
target DNA.
Probes are used to identify which clone of a library
or which band on a gel contains the target DNA.

Southern blotting is a technique for detecting
specific DNA fragments in a complex
mixture. The technique was invented in mid-
1970s by Edward Southern.
Southern blotting is a technique that can detect
mutations in DNA.
It combines the use of
restriction enzymes and
DNA probes.

This technique is capable of detecting a single
fragment in the highly complex mixture of
fragments produced by cleavage of the entire
human genome with a restriction enzyme.
In such a complex mixture, many fragments will
have the same or nearly the same length and
thus migrate together during electrophoresis.
Even though all the fragments are not separated
completely by gel electrophoresis, an
individual fragment within one of the bands
can be identified by hybridization to a specific
DNA probe.

VII- POLYMERASE
CHAIN REACTION

The polymerase chain reaction (PCR) is
a test tube method for amplifying a selected DNA
sequence that does not rely on the biologic
cloning method.
PCR permits the synthesis of millions of copies of
a specific nucleotide sequence in a few hours.
It can amplify the sequence, even when the
targeted sequence makes up less than one part in a
million of the total initial sample.
The method can be used to amplify DNA
sequences from any source-bacterial, viral, plant,
or animal.

1. Primer construction:
It is not necessary to know the nucleotide sequence of
the target DNA in the PCR method.
However, it is necessary to know the nucleotide sequence
of short segments on each side of the target DNA.
flanking sequences
bracket the DNA sequence of interest.
The nucleotide sequences of the flanking regions are
used to construct two single-stranded
oligonucieotides,
usually 20 to 35 nucleotides long,
which are complementary to the respective flanking
sequences.
These synthetic oligonucleotides function as
primers in PCR reactions.

2.Denature the DNA at 94 Cº :
The DNA to be amplified is heated to separate the double-
stranded target DNA into single strands.
3. Annealing of primers to single-stranded DNA at 54 Cº :
The separated strands are cooled and allowed to anneal to
the two primers (one for each strand).
4. Chain extension at 72 Cº :
DNA polymerase and deoxyribonucleoside triphosphates
(in excess) are added to the mixture to initiate the
synthesis of two new chains complementary to the
original DNA chains.
DNA polymerase adds nucleotides to the 3'-hydroxyl end
of the primer, and strand growth extends across the
target DNA, making complementary copies of the
target.

The cycling reactions :
There are three major steps in a PCR, which are repeated for 30 or 40 cycles. This is done on
an automated cycler, which can heat and cool the tubes with the reaction mixture in a
very short time.
1. Denaturation at 94°C :
During the denaturation, the double strand melts open to single stranded DNA, all
enzymatic reactions stop (for example : the extension from a previous cycle).
2. Annealing at 54°C :
The primers are jiggling around, caused by the Brownian motion. Ionic bonds are
constantly formed and broken between the single stranded primer and the single
stranded template. The more stable bonds last a little bit longer (primers that fit
exactly) and on that little piece of double stranded DNA (template and primer), the
polymerase can attach and starts copying the template. Once there are a few bases built
in, the ionic bond is so strong between the template and the primer, that it does not
break anymore.
3. extension at 72°C :
This is the ideal working temperature for the polymerase. The primers, where there are
a few bases built in, already have a stronger ionic attraction to the template than the
forces breaking these attractions. Primers that are on positions with no exact match, get
loose again (because of the higher temperature) and don't give an extension of the
fragment.
The bases (complementary to the template) are coupled to the primer on the 3' side (the
polymerase adds dNTP's from 5' to 3', reading the template from 3' to 5' side, bases are
added complementary to the template)

 At the completion of one cycle of replication, the reaction
mixture is heated again to denature the DNA strands
Each DNA strand ( are now four) binds a complementary
primer, and the cycle of chain extension is repeated.
 By using a heat-stable DNA polymerase (for example, Taq
polymerase) from a bacterium that normally lives at high
temperatures (a thermophilic bacterium),
 the polymerase is not denatured and, therefore, does not have
to be added at each successive cycle.
 Typically twenty to thirty cycles are run during this process,
amplifying the DNA by a million-fold to a billion-fold..
Thus, each newly synthesized polynucleotide can act as a
template for the successive cycles.

Ethidium bromide-stained PCR products after gel electrophoresis. Two sets of primers
were used to amplify a target sequence from three different tissue samples. No
amplification is present in sample #1; DNA bands in sample #2 and #3 indicate
successful amplification of the target sequence. The gel also shows a positive control,
and a DNA ladder containing DNA fragments of defined length for sizing the bands in the
experimental PCRs.

Advantages of PCR
The major advantages of PCR over cloning as a
mechanism for amplifying a specific DNA
sequence are
 sensitivity and speed.
DNA sequences present in only trace amounts
can be amplified to become the predominant
sequence.

Applications
PCR has become a very common tool for a large number of
applications.
1. Comparison of a normal cloned gene with an uncloned mutant form
of the gene: PCR allows the synthesis of mutant DNA in sufficient
quantities for a sequencing protocol without laboriously cloning the
altered DNA.
2. Detection of low-abundance nucleic acid sequences PCR offers a
rapid and sensitive method for detecting viral DNA sequences even
when only a small proportion of cells is harboring the virus.
3. Forensic analysis of DNA samples: DNA fingerprinting by means of
PCR has revolutionized the analysis of evidence from crime scenes.
DNA isolated from a single human hair, a tiny spot of blood, or a
sample of semen is sufficient to determine whether the sample
comes from a specific individual.
4. Prenatal diagnosis and carrier detection of cystic fibrosis: Cystic
fibrosis is an autosomal recessive genetic disease resulting from
mutations in the cystic fibrosis transmembrane regulator (CFTR)
gene.

The tools of biotechnology not only allow
the study of gene structure, but also
provide ways of analyzing the products
of gene expression.
A.Determination of mRNA levels
B. Analysis of proteins

A. Determination of mRNA levels
Messenger RNA levels are usually determined by
the hybridization of labeled probes to either
mRNA itself or to cDNA produced from mRNA.
1. Northern blots:
is a technique used in molecular biology research to
study gene expression by detection of RNA (or
isolated mRNA) in a sample then transferred to a
membrane and hybridized to a radio active
probe.
The bands obtained by autoradiography give a
measure of the amount and size of particular
mRNA molecules in the sample.

In the Southern blotting, DNA fragments are
denatured with alkaline solution. In the
Northern blotting, RNA fragments are treated
with formaldehyde to ensure linear
conformation.

The steps in northern blotting include:
• RNA isolation
• Gel electrophoresis of RNA for separation
• Transfer to membrane (usually positively
charged nylon as RNA is negatively charged)
• Cross-linking of RNA to membrane (usually
by UV-crosslinking or chemical means)
• Hybridization
• Detection

The northern blot protocol and its
variations are used however in molecular
biology research to:
• a gold-standard for the direct study of gene
expression at the level of mRNA (messenger
RNA transcripts).
• detection of mRNA transcript size
• study RNA degradation
• study RNA splicing - can detect alternatively
spliced transcripts
• study RNA half-life
• study IRES (internal ribosomal entry site).

2. Microarrays: DNA microarrays contain thousands of
immobilized DNA sequences organized in an area no larger
than a microscope slide.
These microarrays are used to analyze a sample for:
1. For genotyping analysis
2. DNA microarrays are often used to determine the differing
patterns of gene expression in two different types of cell-
for example, normal and cancer cells.
 The population of mRNA molecules from a particular
cell type is converted to cDNA and labeled with a
fluorescent tag.
 This mixture is then exposed to a gene chip, which is a
glass slide or membrane containing thousands of tiny spots
of DNA, each corresponding to a different gene.
The amount of fluorescence bound to each spot is a measure
of the amount of that particular mRNA in the sample.

Principle
 The core principle behind microarrays is
hybridization between two DNA strands.
 A high number of complementary base pairs in a
nucleotide sequence means tighter non-covalent
bonding between the two strands. After washing off of
non-specific bonding sequences,
only strongly paired strands will remain hybridized.
 So generate a signal that depends on the strength of the
hybridization determined by the number of paired
bases.
 Microarrays use relative quantization in which the
intensity of a feature is compared to the intensity of the
same feature under a different condition, and the
identity of the feature is known by its position.

Gene expression values
from microarray
experiments can be
represented as heat maps
to visualize the result of
data analysis

B. Analysis of proteins
When investigating one, or a limited number of
gene products, it is convenient to use labeled
antibodies to detect and quantify specific
proteins. However, when analyzing the
abundance and interactions of large numbers
of cellular proteins, automated methods
employing two-dimensional gel
electrophoresis, mass spectrometry,
multidimensional liquid chromatography, and
bioinformatics are employed.

1. Enzyme-Linked Immunosorbent
Assays (ELlSAs):
ELISA, an unknown amount of antigen is
affixed to a surface, and then a specific
antibody is applied over the surface so that it
can bind to the antigen.
This antibody is linked to an enzyme, and in
the final step a substance is added that the
enzyme can convert to some detectable
signal, most commonly a colour change in a
chemical substrate.

Applications
Because the ELISA can be performed to evaluate
either the presence of antigen or the presence
of antibody in a sample, it is a useful tool for
determining serum antibody concentrations
(such as with the HIV test or West Nile Virus).
It has also found applications in the food
industry in detecting potential food allergens
such as milk, peanuts, walnuts, almonds, and
eggs. ELISA can also be used in toxicology as
a rapid presumptive screen for certain classes
of drugs.

2. Western blots: (Immunoblots )
The Western blot ( protein immunoblot) is a
widely used analytical technique used to detect
specific proteins in the given sample of tissue
homogenate or extract
 Protein molecules in the sample are separated
by electrophoresis and blotted to a membrane.
 The probe is a labeled antibody, which
produces a band at the location of its antigen.

Medical diagnostic applications
• The confirmatory HIV test employs a Western
blot to detect anti-HIV antibody in a human
serum sample.
• A Western blot is also used as the definitive test
for Bovine spongiform encephalopathy (BSE,
commonly referred to as 'mad cow disease').
• Some forms of Lyme disease testing employ
Western blotting.
• Western blot can also be used as a confirmatory
test for Hepatitis B infection.
• In veterinary medicine, Western blot is sometimes
used to confirm FIV+ status in cats

3. Proteomics :
Proteomics involves the systematic study of proteins in
order to provide a comprehensive view of the structure,
function and regulation of biological systems.
Proteome
Two popular definitions:
• All the proteins that can be synthesized by the
cell.
• All the proteins synthesized by a particular cell
at a particular time.
Clinical research also hopes to benefit from
proteomics by both the identification of new
drug targets and the development of new
diagnostic markers.

How To Study?
1. Isolate a homogeneous population of cells
(e.g., yeast cells that have just been switched from
glucose to galactose as their energy source).
2. Extract the contents of the cells and separate the
mix of proteins from other components.
3. Separate the proteins in the mix by
two-dimensional (2D) gel electrophoresis.
This separates the proteins
– in one dimension by their electrical charge;
– in the second dimension by their size.
4. Stain the gel to visualize the various spots of
protein.

5. Punch out a spot.
6. Add a protease to digest the protein in that
spot into a mix of peptides.
7. Run the mix through a mass spectrometer,
which will separate the peptides into sharply-
defined peaks.
8. Run the resulting data through a database of
all known proteins (that have been digested
with the same enzyme) to see if you can find a
match.

Gene therapy is a technique for correcting
defective genes responsible
for disease development.
Researchers may use one of several approaches
for correcting faulty genes:
Replacing a mutated gene that causes disease
with a healthy copy of the gene.
Inactivating, or “knocking out,” a mutated
gene that is functioning improperly.
Introducing a new gene into the body to help
fight a disease.

How does gene therapy work?
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.
Some of the different types of viruses used as
gene therapy vectors:
Retroviruses , Adenoviruses , Adeno-associated
viruses - and Herpes simplex viruses.

Vector is a DNA molecule used as a vehicle to
transfer foreign genetic material into another
cell.
o The four major types of vectors are
plasmids, viruses, cosmids (hybrid plasmid),
and artificial chromosomes.
Insertion of a vector into the target cell is usually
called
 transformation for bacterial cells,
 transfection for eukaryotic cells,
 transduction insertion of a viral vector

Besides virus-mediated gene-delivery systems, there are
several nonviral options for gene delivery.
 The simplest method is the direct introduction of
therapeutic DNA into target cells.
This approach is limited in its application because it can
be used only with certain tissues and requires large
amounts of DNA.
 The creation of an artificial lipid sphere with an
aqueous core. This liposome, which carries the
therapeutic DNA, is capable of passing the DNA
through the target cell's membrane.
 Therapeutic DNA also can get inside target cells by
chemically linking the DNA to a molecule that will
bind to special cell receptors. Once bound to these
receptors, the therapeutic DNA constructs are engulfed
by the cell membrane and passed into the interior of the
target cell. This delivery system tends to be less
effective than other options.

There are other gene therapy techniques, although
they aren’t as frequently used.
 One method involves inserting therapeutic DNA
into cultured endothelium tissue (endothelium is
the membrane that lines all of the blood vessels)
and then grafting it into the patient.
Another technique requires the patient to receive
an electric shock while submerged in a bath of a
therapeutic DNA solution. The shock opens the
skin pores, allowing the DNA to enter. Still other
options include skin grafts, connective tissue
grafts, and injecting the liver with the therapeutic
DNA.

 Chemicals called restriction enzymes act as the scissors
to cut the DNA.
Thousands of varieties of restriction enzymes exist, each
recognizing only a single nucleotide sequence. Once it
finds that sequence in a strand of DNA, it attacks it and
splits the base pairs apart, leaving single helix strands at
the end of two double helixes.
Scientists are then free to add any genetic sequences they
wish into the broken chain and, afterwards, the chain is
repaired (as a longer chain with the added DNA) with
another enzyme called ligase.
Hence, any form of genetic material can be spliced
together; bacteria and chicken DNA can, and have
been, combined. More often, though, splicing is used
for important efforts such as the production of insulin
and growth hormone to cure human maladies.

Another aspect of gene therapy is gene silencing,
also called antisense technology:
Interruption or suppression of the
expression of a gene at transcriptional or
translational levels.
With this method, geneticists can inactivate a
gene that may cause disease or be defective.
Gene silencing is used to treat several viruses
including AIDS, Herpes, Chicken Pox, and
Hepatitis. More importantly, though, antisense
technology is used by geneticists in research to
learn what happens when certain genes are
silenced.

Comparison of gene silencing strategies

Many disorders of the brain and nervous system are caused, at
least in part, by genes that create detrimental proteins, detrimental
protein accumulation, or detrimental protein activity.
To counter this negative side of genes, researchers recently
developed a method, known as RNA interference, that
straightjackets or "silences" select genes and significantly reduces
production of problematic proteins.

What factors have kept gene therapy from becoming
an effective treatment for genetic disease?
 Short-lived nature of gene therapy - Before gene therapy can become a
permanent cure for any condition, the therapeutic DNA introduced into
target cells must remain functional and the cells containing the therapeutic
DNA must be long-lived and stable. Problems with integrating therapeutic
DNA into the genome and the rapidly dividing nature of many cells prevent
gene therapy from achieving any long-term benefits. Patients will have to
undergo multiple rounds of gene therapy.
 Immune response - Anytime a foreign object is introduced into human
tissues, the immune system is designed to attack the invader. The risk of
stimulating the immune system in a way that reduces gene therapy
effectiveness is always a potential risk. Furthermore, the immune system's
enhanced response to invaders it has seen before makes it difficult for gene
therapy to be repeated in patients.

 Problems with viral vectors –
Viruses, while the carrier of choice in most gene therapy
studies, present a variety of potential problems to the patient
--toxicity, immune and inflammatory responses, and gene
control and targeting issues. In addition, there is always the
fear that the viral vector, once inside the patient, may
recover its ability to cause disease.
 Multigene disorders –
Conditions or disorders that arise from mutations in a single
gene are the best candidates for gene therapy. Unfortunately,
some the most commonly occurring disorders, such as heart
disease, high blood pressure, Alzheimer's disease, arthritis,
and diabetes, are caused by the combined effects of
variations in many genes. Multigene or multifactorial
disorders such as these would be especially difficult to treat
effectively using gene therapy. For more information on
different types of genetic disease,

What are some recent developments in gene therapy research?
The FDA approves clinical trials of the use of gene therapy on thalassemia major patients in the US.
Researchers at Memorial Sloan Kettering Cancer Center in New York begin to recruit 10 participants
for the study in July 2012. The study is expected to end in 2014.
In March 2013, Researchers at the Memorial Sloan-Kettering Cancer Center in New York, reported that
three of five subjects who had acute lymphocytic leukemia (ALL) had been in remission for five
months to two years after being treated with genetically modified T cells which attacked cells with
CD19 genes on their surface, i.e. all B-cells, cancerous or not. The researchers believed that the
patients immune systems would make normal T-cells and B-cells after a couple of months however
they were given bone marrow to make sure. One patient had relapsed and died and one had died of a
blood clot unrelated to the disease.
Successful Transplant of Patient-Derived Stem Cells Into Mice With Muscular Dystrophy (June 27,
2012) — Stem cells from patients with a rare form of muscular dystrophy have been successfully
transplanted into mice affected by the same form of dystrophy, according to a new
In July 2012, the European Medicines Agency recommended approval of a gene therapy treatment for
the first time in either Europe or the United States. The treatment, called Glybera, compensates for
lipoprotein lipase deficiency, which can cause severe pancreatitis. The recommendation was
endorsed by the European Commission in November 2012 and commercial rollout is expected in late
2013.
In December 2012, it was reported that 10 of 13 patients with multiple myeloma were in remission "or
very close to it" three months after being injected with a treatment involving genetically engineered
T cells to target proteins NY-ESO-1 and LAGE-1 which exist only on cancerous myeloma cells. This
procedure had been developed by a company called Adaptimmune.

Genome Editing -- A Next Step in Genetic Therapy -- Corrects
Hemophilia in Animals ScienceDaily (June 26, 2011) — Using an
innovative gene therapy technique called genome editing that hones in on
the precise location of mutated DNA, scientists have treated the blood
clotting disorder hemophilia in mice. This is the first time that genome
editing, which precisely targets and repairs a genetic defect, has been done
in a living animal and achieved clinically meaningful results.
Bio-Engineered Protein Shows Promise as New Hemophilia Therapy (Oct.
27, 2011) — A genetically engineered clotting factor that controlled
hemophilia in an animal study offers a novel potential treatment for human
hemophilia and a broad range of other bleeding problems
New DNA Repair Pathway (Nov. 13, 2010) — Researchers have found a
new, inducible pathway for repairing DNA damaged by oxygen
New Animal Model for Hemophilia A Developed (Sep. 4, 2010) —
Researchers have developed a new animal model for studying hemophilia
A, with the goal of eventually treating people with the disorder.
.

• Broad Therapy For Muscular Dystrophy (June 24, 2009) — Scientists have demonstrated that the
glycosyltransferase Galgt2 can lessen symptoms in multiple models of muscular dystrophy.
• Nanotechnology + gene therapy yields treatment to torpedo cancer. March, 2009. The School of
Pharmacy in London is testing a treatment in mice, which delivers genes wrapped in nanoparticles to
cancer cells to target and destroy hard-to-reach cancer cells
• Results of world's first gene therapy for inherited blindness show sight improvement. 28 April
2008. UK researchers from the UCL Institute of Ophthalmology and Moorfields Eye Hospital NIHR
Biomedical Research Centre have announced results from the world’s first clinical trial to test a
revolutionary gene therapy treatment for a type of inherited blindness. The results, published today
in the New England Journal of Medicine, show that the experimental treatment is safe and can
improve sight. The findings are a landmark for gene therapy technology and could have a significant
impact on future treatments for eye disease.
• Hurdles For Muscular Dystrophy Therapy Cleared (Oct. 29, 2008) — Boys with Duchenne
Muscular Dystrophy will usually lose the ability to walk by their teens and typically die before the
age of 30.
• Researchers Discover Molecular Basis Of A Form Of Muscular Dystrophy (Apr. 30, 2008) —
Researchers report that people with limb-girdle muscular dystrophy are missing a protein called c-
FLIP, which the body uses to prevent the loss of muscle tissue.
• Massive MicroRNA Scan Uncovers Leads To Treating Muscle Degeneration (Oct. 17, 2007) —
An increasing number of genes have been linked to muscular dystrophy and related disorders that
cause muscle weakness and wasting, but it's still largely unknown how these genes cause disease,
and,

STUDY QUESTIONS
 Define the following terms:
 [Billion, Crystallography, X-ray diffraction, neutron diffraction, electron diffraction,
Chromatography, electrophoresis electropherogram ultracentrifugation, Macromolecules,
buoyancy, Spectroscopy, DNA cloning, mutations, Denaturation, Annealing, extension,
hybridization , antigen, immunoblot, Gene therapy, etc]
 Respond to the following questions:
 State and explain all the main reasons for the need of biotechnological knowledge in
pharmaceutics
 State and describe some of the biotechnological methods that are part of biotechnology
knowledge and skills learning in pharmaceutics
 Describe the serial activities of DNA Cloning as part of biotechnological process
 Group work discussional questions:
 With references to the Type, procedural process and basic procedural stages of biotechnological
knowledge and skills, describe the main clinical applications of the following biotechnological
procedures:
 X-ray Diffraction Reveals the Molecular Structure of DNA
 Chromatography Electrophoresis
 Ultracentrifugation
 Southern Blotting
 Polymerase Chain
 Analysis of gene expression
 Describe the various conventional ways the biotechnological knowledge and skills can be applied
to modernize the traditional pharmaceutical procedures such as dosage form formulations, drug
molecular carriers and delivery, drug administration, storage, etc.

7 biotechnology and human disease

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