1. GENETIC DISEASES

2. Gene Mutations
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Deletion, reading frame shifts
Substitution, one base
replaced by another
Duplication, repetition of part
of the sequence
Addition, Addition extra base
Change in one or more
nucleotide bases in the DNA
Change in the genotype (may
be inherited)

3. Cystic fibrosis
• People with cystic fibrosis inherit a
defective gene on chromosome 7
called CFTR (cystic fibrosis
transmembrane conductance
regulator). Mutation causes the
deletion of 3 bases in DNA
resulting in one amino acid
(phenylalanine) not being coded
for

5. • The protein produced by this gene normally helps salt
(sodium chloride) move in and out of cells. If the protein
doesn't work correctly, that movement is blocked and an
abnormally thick sticky mucous is produced on the
outside of the cell.

6. •
•
The cells most seriously affected by this are the lung cells. This
mucous clogs the airways in the lungs, and increases the risk of
infection by bacteria.
The thick mucous also blocks ducts in the pancreas, so digestive
enzymes can't get into the intestines. Without these enzymes, the
intestines cannot properly digest food. People who have the
disorder often do not get the nutrition they need to grow normally.

7. Cystic Fibrosis - Defective Gene
• Mutation causes the deletion of 3 bases in DNA.
One amino acid (phenylalanine) is not coded for
in the
Cystic Fibrosis Transmembrane Regulator
CFTR protein
• Faulty CFTR protein cannot control the opening
of chloride channels in the cell membrane
• Results in production of thick sticky mucus,
especially in lungs, pancreas and liver
• Organs cannot function normally and infection
rate increases

8. These are bacteria, Pseudomonas aeruginosa, a human pathogen that
is a particular problem in the lungs of cystic fibrosis patients

9. Phenylketonuria (PKU) – Defective Gene
•
NORMAL – Melanin pigment produced

10. PKU
•
Anna with her children Madeleine (centre), who has PKU, and Isobel.
Madeleine's PKU is managed successfully by diet.
www.sciencemuseum.org.uk/exhibitions/genes

11. PKU
1.
Gene mutation in DNA coding for the enzyme phenylalanine
hydroxylase
2.
Phenylalanine hydroxylase not produced
3.
Amino acid phenylalanine cannot be converted to the amino acid
tyrosine
4.
Tyrosine is necessary to produce the pigment melanin

12. 5.
6.
7.
Phenylalanine collects in the blood and causes retardation in young
children
Managed by controlling diet to eliminate proteins containing
phenylalanine
Disease is tested by drops of blood taken from the baby

13. Genetic screening and DNA
probes
• Genetic screening detects particular
genes or chromosome mutations (e.g.
cystic fibrosis, …)
• DNA probes are used to diagnose
diseases caused by defective genes or
oncogenes
• Need to know specific base sequence
found in defective gene

14. Summary
•
DNA extraction (e.g. white
blood cells, gametes)
•
Cut the DNA at gene loci with
restriction enzymes
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Split DNA fragments up on the
basis of their size with
electrophoresis gel
•
Southern blotting and use of
radioactive DNA probe to
locate the fragments of DNA
•
Autoradiography to create an
image of the DNA pattern

15. Stage 1 – DNA extraction
• Small sample of tissue (e.g. blood) is
mixed with water-saturated phenol and
chloroform
• Causes proteins to precipitate out leaving
DNA in the water layer
• DNA can now be extracted from the water
layer and purified

16. Stage 2 – Restriction enzymes
• Each restriction enzyme is
specific to one base
sequence
• Cut the DNA (cleavage)
after enzymes have
attached to all recognition
sites
• Fragments produced are
called restriction fragment
length polymorphisms
(RFLPs)
• Some produce blunt ends,
some sticky ends (more
useful)

17. Single stranded complementary DNA strand
(cDNA) is labelled with an radioactive isotope

18. Stage 3 – Electrophoresis
• Electrophoresis separates DNA fragments
according to their size and electrical charge
• DNA mixture is placed in a well at one end of a
gel (made of agarose)
• Electrical current will move the DNA fragments
to the positively charged electrode
• Phosphate is highly positive, making nucleotide
negative

19. Stage 4 – Southern Blotting and DNA
probes
• Heat DNA on the gel to unwind and make single
stranded DNA
• A nylon membrane placed over the gel is covered with
absorbent paper / single stranded fragments are
transferred to membrane by capillary action
• Fix fragments on membrane with UV light
• Put membrane into solution containing the DNA probe
• DNA probe attaches to complementary base sequences
of the disease-causing gene / fragment is labelled
radioactive

21. Stage 5 – Autoradiography
• Radioactive solution is washed off and an X-Ray
plate is placed over the membrane
• Radioactive probes (32p) will give off radiation
causing a pattern of bands on the X-ray plate,
conforming the presence of the disease causing
gene
• Mutant gene is missing a restriction site which is
present at normal genes
– Mutant gene will travel shorter distances than normal
DNA

23. Southern blot
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The first process of the Southern blot is agarose gel electrophoresis, a technique that
separates DNA fragments by size.
A mixture of DNA is digested with restriction enzymes, which cut the DNA into small
pieces of various sizes, and placed into different lanes of an agarose gel.
An electric current, which is run through the gel, forces the DNA fragments to move
through the agarose matrix. Next, the size-fractionated DNA, while still on the gel, is
chemically denatured and blotted onto a sheet of nitrocellulose paper.
The nitrocellulose is then heated so that the single stranded DNA fragments are
permanently adhered to its surface. The Southern blot is now ready to be analyzed.
A radioactive single stranded probe is hybridized with the DNA in question. The probe
anneals to the DNA wherever a complementary sequence of genetic code exists.
After the radioactive probe has been allowed to bond with the denatured DNA on the
nitrocellulose paper, an X-ray is taken of the nitrocellulose sheet. Only the areas where
the probe binds to the denatured DNA will show up on the X-ray film. This allows
researchers to identify the spatial location and frequency of the specific genetic
sequence contained by the probe (Brinton and Lieberman, 1994).