Transcript Section 9 – Human therapeutics and forensic uses

Unit 1
Cell and Molecular
Biology
Section 9
Human therapeutics and forensic
uses
What is DNA?
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DNA is the carrier of genetic information and
provides a structural plan for proteins.
It consists of linear linked nucleotides whose
sequence forms hereditary.
The DNA is in the form of a double helix and
is made up of four bases: adenine, thymine,
cytosine and guanine.
DNA Uses for Forensic
Identification
DNA is used in forensics to:
 Identify potential suspects if their DNA matches DNA found at
crime scene
 Prove possible innocence of people wrongly accused of crime
 Identify crime and catastrophe victims
 Show paternity and other family relationships
 Identify endangered/protected species (can be used to prosecute
poachers)
 Detect bacteria polluting air, soil, water, food
 Match organ donors with organ receivers
 Determine pedigree
Solving Crimes
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DNA can be used to identify criminals with
incredible accuracy when biological evidence
exists.
Still not used to convict people for a long time
as juries didn’t understand how the DNA
evidence proved anything.
Samples could be contaminated easily.
DNA Fingerprinting
This works on the principle that individuals poccess regions
within their chromosomes that have short repeat sequences
The number of times a sequence repeats is unique to each
individual
These regions are known as minisatellite regions with the
repeats being known as variable number tandem repeats VNTR
In forensics specific enzymes are used to cut the DNA at these
regions and the fragments separated by electrophoresis
Since every person has a unique number and pattern of VNTR
they will produce a DNA fragments which vary in number and
size – this shows up as a unique banding pattern
Who is the Daddy?
In this example a single restriction enzyme has cut the
DNA at specific points generating a small number of
fragments.
M
1
children
2
3
4
F
paternal
bands
maternal
bands
DNA
profiling:
Singleprobe
locus
Who is the Baddy?
In this example a variety of restriction enzymes have
cut the DNA at many specific points generating a
large number of fragments.
DNA
profiling:
Multi-locus
probe
Southern Blot Technique
DNA fingerprinting uses a technique known as southern
hybridisation and also involves genetic probes to target stable
areas on either side of the minisatellite regions. There are six
main steps
1.
DNA extraction and amplification of sample
2. Digestion of DNA with a restriction endonuclease
3. Agarose gel electrophoresis
4. Preparation of a "southern blot"
DNA fragments are transferred to the surface
of a nylon membrane by blotting. This
denaturation/blotting procedure is known as a
"Southern Blot" .
The blotting of DNA to a nylon membrane
preserves the spatial arrangement of the DNA
fragments that existed after electrophoresis.
5. The southern blot is hybridised with a
radioactive probe. Probes are
radioactive sections of DNA or RNA with
a base sequence complementary to the
region being targeted. In this case a
single locus on one chromosome
6. Autoradiography - Pressing the
southern blot onto photographic paper
shows the regions bound to the probe
as black lines or blots.
Cystic Fibrosis (CF)
The CF gene codes for a membrane carrier protein present in
epithelial cells called CF transmembrane conductance
regulator (CFTR). This controls the passage of chloride ions
across the cell membrane.
In patients with cystic fibrosis the structure of the protein is
altered resulting in problems with the flow of chloride and
sodium ions into and out of the cell across the membrane.
The lung is the main organ affected by CF. If the chloride
channel is blocked the airway becomes drier. This causes
sticky mucus to build up in the lungs.
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Major symptoms: inflammation of lung tissue and
persistent bacterial infection.
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Other symptoms: defects in the pancreas.
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The disease is relatively easy to diagnose as a
symptom is the production of salty sweat.
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Affected individuals have a reduced quality of
life and a life expectancy of about 30 years.
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Daily physiotherapy and drugs are used to rid
the lungs of the build up of mucus.
Diseases caused by a single-gene defect
are known as monogenic traits and are
characterised as either:
1. Autosomal dominant
2. Autosomal recessive
3. X-linked (sex-linked) which are
mostly recessive
Diseases that involve several genes are
known as polygenic traits and they are
usually more difficult to diagnose and
treat than the single-gene defects.
Cysti fibrosis – case study
CF shows a simple Mendelian inheritance pattern
as it is a an autosomal recessive monogenic trait
(mutation).
Cystic fibrosis is caused by a single faulty gene
NORMAL = Cf
ABNORMAL = Cf
We each carry TWO alleles for the Cf gene, thus:
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NORMAL
= Cf Cf
CARRIER
= Cf Cf
AFFECTED = Cf Cf
Cystic fibrosis appears when two carriers
produce a child:
Mother
Cf
Cf
Cf Cf
Cf Cf
Cf
Father
Cf
The gene linked to CF was identified on chromosome 7.
band
7q22
chromosome 7
physical markers
0
100
CF
200
300
400
280 kb contig
1
2
3
4
?
CFTR gene
500 kbp
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There are over 700 different mutations that have been
identified in the CFTR gene which helps, in part, to explain
why different CF patients are affected to different degrees.
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The variation in how the disease is expressed in the
phenotype may also be due to environmental factors and
the effect of modulator genes on the CFTR gene.
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During genetic screening 12 of the mutations are currently
tested for, one of which is ΔF508. Δ indicates that it is a
deletion mutation, F is phenylalanine and 508 is the
position of the deletion in the protein. The ΔF508 mutation
accounts for 70% of CF cases worldwide.
(see the following two slides)
The cystic fibrosis DF508 mutation (1)
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Diagram how transcription produces the primary RNA transcript.
This is converted into the functional mRNA by removal of
intervening sequences.
On translation the CF transmembrane conductance regulator
protein (CFTR) is produced.
The cystic fibrosis DF508 mutation (2)
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Diagram shows the normal and mutated proteins.
Normal CFTR has phenylalanine (F) at position 508.
In the mutant form this is deleted, causing the protein
to fold incorrectly.
Test for cystic fibrosis defective allele
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A short sequence that spans the mutated region is amplified using
the polymerase chain reaction.
In lane 1 a normal homozygous pattern is shown.
In lane 2 a carrier (hererozygous) shows two bands, one normal
and one smaller by 3 nucleotides (one codon, representing the
deleted phenylalanine).
In lane 3 a CF-affected individual (homozygous recessive) shows
one band at the lower position.
Duchenne’s Muscular
Dystrophy (DMD)
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Duchenne’s muscular dystrophy is a X-linked disease that
affects around 1 in 3,300 boys.
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It causes progressive wasting of the muscles so that by the
time the person is in their teens they are likely to be
confined to a wheelchair.
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The gene was identified in 1987. The gene codes for the
protein dystrophin.
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Dystrophin’s normal function is to link the cytoskeleton with
the sarcolemma (muscle cell membrane) in muscle cells.
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Life expectancy is similar to CF, i.e. about 30 years.
Human Therapeutics
The techniques discussed in the previous lessons
are now used to detect genetic disorders such as
cystic fibrosis and Duchenne’s muscular dystrophy.
This can lead to the development of a screening test
which must be used in conjunction with counselling.
Congenital abnormalities are genetically-based
diseases (often simply called genetic diseases)
that are present at birth.
Cystic fibrosis and Duchenne’s muscular dystrophy are two
examples of genetic diseases where molecular genetics has
led to improved diagnosis and treatment.
Genetic diseases are first recognised by the disease
symptoms and there are a number of steps required to
establish the definitive genetic cause:
1.Trace the disease through family relationships by carrying out
pedigree analysis to determine if the faulty gene is
dominant, recessive or X-linked.
2.Once the disease has been identified as a monogenic trait then
a search for the gene defect can take place using the
processes similar to those employed for the human genome
project, i.e.
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Genetic mapping – by looking for genetic markers that
are co-inherited with the disease. The more often the
faulty gene and marker are co-inherited, the closer they
are on the chromosome.
Physical mapping
DNA sequencing
Cystic fibrosis: pedigree
analysis
CF shows a simple Mendelian inheritance pattern
as it is a an autosomal recessive monogenic trait
(mutation).
Cystic fibrosis is caused by a single faulty gene
NORMAL = Cf
ABNORMAL = Cf
We each carry TWO alleles for the Cf gene, thus:
NORMAL
= Cf Cf
CARRIER = Cf Cf
AFFECTED = Cf Cf
Cystic fibrosis appears when two carriers
produce a child:
Mother
Cf
Cf
Cf
Cf Cf
Cf Cf
Cf
Cf Cf
Cf Cf
Father
Searching for the mutated
gene
The gene was eventually identified in 1989
by examining cloned DNA fragments
using the techniques of chromosome
walking and chromosome jumping shown
on the following two slides
Often a mixture of ‘walks’ and ‘jumps’ is needed
to progress as shown in the diagram above.
Chromosome walking
In chromosome walking the end of each overlapping
fragment is used in a hybridisation test to identify the
next fragment.
This is often used to ‘walk’ from a marker gene
towards the target gene.
Chromosome walking
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Chromosome jumping is a similar technique to
chromosome walking but in this case a special cloning
technique is used to isolate complementary fragments
that are far apart.
This enables a ‘jump’ along the chromosome which is
useful if the marker gene is far from the target gene.
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Using the three techniques of genetic mapping,
physical mapping, genetic mapping and DNA
sequencing have allowed the position and sequence
of the mutation to be identified.
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Once this information is known it is possible to
produce a tests which can identify the mutation if
present in an individual. This involves the use of
hybridised DNA and gene probes
Steps
 DNA sample taken from patient, cut with restriction
enzymes and run through electrophoresis gel
 The gel is blotted using the Southern Blot
 The DNA is hybridised with a radioactive probe – this
binds to the mutated gene
 Autoradiography shows the presence of the mutated
gene
Testing for the CF allele
The 3-bp deletion:
Normal gene sequence
Mutant gene sequence
Ile
Ile Phe Gly
5' - GAA AAT ATC ATC TTT GGT GTT TCC - 3'
5' - GAA AAT ATC ATT GGT GTT TCC - 3'
Ile Ile Gly
Amplify this region using PCR:
Use primers about 100 bp apart
Normal allele produces 100 bp fragment, mutant a 97 bp fragment
A PCR-based test is used to identify the
cystic fibrosis (CF) defective allele. After
amplifying a short DNA fragment which
spans the area of the deletion, the DNA
fragments are then separated on an
electrophoresis gel. (see the following
slide)
NOTE: In lanes 1 and 3, shown on the
following slide, the DNA band contains
sequences from both paternal and
maternal chromosomes that run to the
same position in the gel. It is only in the
heterozygous case that the two bands are
distinguished.
Test for cystic fibrosis
defective allele
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A short sequence that spans the mutated region is amplified
using the polymerase chain reaction.
In lane 1 a normal homozygous pattern is shown.
In lane 2 a carrier (hererozygous) shows two bands, one
normal and one smaller by 3 nucleotides (one codon,
representing the deleted phenylalanine).
In lane 3 a CF-affected individual (homozygous recessive)
shows one band at the lower position.
Gene therapy for cystic
fibrosis
(a) In vivo gene therapy
insert transgene
into liposomes or
lipoplexes, or viral
vector/vehicle systems
(b) Ex vivo gene therapy
deliver to site of
action (e.g. lung) by
aerosol spray
remove cells
grow cells ex vivo
add transgene and
select modified cells
replace the
modified cells
Human therapeutics – Gene
therapy
1.
The possibility of replacing a defective gene with a
‘good’ copy of the gene to overcome the problems
caused by the defect is called gene therapy. In this
way one is able to deal with the cause not just the
symptoms.
2.
Gene therapy might also be used to kill abnormal
cells, such as cancerous cells, or to inhibit the
spread of viruses by preventing DNA replication.
3.
In the case of cancer, gene therapy would avoid many
of the side-effects associated with the drugs used in
chemotherapy.
4.
The difficulty with gene therapy in practical
terms is that there are difficult criteria to be met
before it can be used. The conditions are:
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the defective gene function is characterised;
the normal gene is available in cloned form;
the affected cells are accessible, either the
cells can be treated outside the body (ex vivo)
and replaced, or may have to be treated
within the patient’s body (in vivo);
there are suitable vehicles (vectors) for
delivery of the gene e.g. viruses, liposomes or
artificial chromosomes
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viral vectors – virus have evolved to alter a host cells
metabolism by implanting their DNA into the host cells DNA.
Altered viruses with therapeutic genes can be given to patient
with virus implanting therapeutic gene. ( risky)
the use of liposomes that can fuse with cell membranes
human artificial chromosomes - the gene functions normally
in its target cells.
The difficulty of meeting the criteria means that gene therapy is still
in the early stages of development although progress is being
made.
Gene therapy which raises ethical questions in the type of cells that
are the targets, i.e. whether somatic cells (body cells) or germ
cells are used. If genes within reproductive cells could be altered
then the alteration could be passed on to the next generation and
effectively alter the species gene pool.