Biotechnology


4 major areas
Human Genome Project
 Gene Therapy
 Forensic science
 Agriculture
Human Genome Project

Aim
– Identify sequence of bases on all 23 human
chromosomes (3 billion bases)
– Identify genes within those sequences (~30 000 genes)
– Locate the position of the genes on the chromosomes

$6 billion dollars, 1000 scientists, 50 countries,
completed 2000!
 Only ~3% of genome codes for protein
– Remainder is regulatory or of unknown function (junk)
e.g repetitive sequence, possibly viral DNA
Human Genome Project –
Approach Used

Concept
– Produce ever more detailed maps of chromosomes
– 1. Genetic
linkage
–3. Chop
chromosome
- map (low resolution)
small, overlapping
 Relative order & spacing of disease linked genes (not physical map)
fragments
– 2. Combine with STS/EST (sequence tag site/ expressed
–Sequence
sequence tag) maps
 Position
–Computers
alignof unique DNA sequences (physical map)
 Linkage
data to disease genes
overlapping
sequences
– Pain stakingly slow, but links to useful disease information
– Alternatively SHOTGUN sequencing
Human Genome Project
Methods
Restriction Enzymes Summary

Variety of enzymes

Isolated from bacteria

Cut DNA at specific sequences
 Used to produce DNA fragments
– Blunt or sticky ended

DNA Ligase (not a RE), used to LIGATE
(joins DNA) fragment into a plasmid
 Animation
Pst1
BamHI
11kb
6kb
BamHI
3kb
BamHI
BamHI
1236bp
PvuI
1875 bp
900bp
PvuI
HindIII
1670bp
DNA Amplification

To increase the concentration of specific pieces of DNA
 PCR (polymerse chain reaction)
–
–
–
–
Thermostable Taq DNA polymerase
Nucleotides (AGCT)
template DNA
Primers (bind to DNA, initiate DNA replication)


Either side of area of genome to be amplified
Repeated cycles of heating and cooling
– Heating – breaks apart DNA template
– DNA primers anneal (hydrogen bond) as cools
– DNA polymerase synthesises complementary strand

Video Video 2
What is electrophoresis?

Separation of charged molecules.
 DNA is negatively charged; attracted to the
positive terminal
 Small molecules easily pass through spaces
in gel, so travel faster.
 Larger molecules have difficulty travelling
through spaces in agarose.
so in DNA agarose gel electrophoresis the
fragments are separated by size.
Electrophoresis Gel
Preparation
Molten agarose 55 - 60°C
Comb
Tray
During polymerisation
the sugar molecules
all cross link with
each other causing
the solution to ‘gel’
into a semi-solid
matrix; a bit like jelly
in a trifle!
DNA SIZE MARKERS/STANDARDS
-ve
Smaller fragments travel faster.
 The sizes of bands are known
(in base pairs).

largest
smallest
+ve
Typical DNA gel showing bands of
DNA of different sizes.
First and last lanes contain DNA size markers
DNA sequencing

4 tubes with DNA polymerase, template DNA
– DNA nucleotides
– 1 Dideoxynucleotide (e.g. ddATP, terminates DNA
synthesis where A is located) labelled (radioactive / 4
fluorescent colours)

Produces strands of DNA terminated at different
points
– Fragments separated by electrophoresis
– Labels visualised by autoradiography or computer
(fluorescence)
– VIDEO
DNA probes

Short sequences of DNA complementary to
specific sequences in the genome
– Labelled (radioactive/ fluorescent)
– Binds to complementary sequence

Used extensively
– Search for genes
– Locate genes (FiSH – fluorescence
immunohistochemistry)
– DNA fingerprinting
Using a Probe to Find
Sequences
on
a
Gel
Usually a nitrocellulose membrane
DNA on the gel
is double stranded &
needs to be singlestranded for probe
to bind: gel treated
with sodium hydroxide
to do this
Chromosome Walking

Marker sequence identified
– Target gene is some distance from marker
– 2 Restriction enzymes digest DNA
– Probe to find fragments containing marker DNA
– Sequence 3’ ends
– Probe for these sequences, repeat above
– Use overlaps in digests to identify fragment order
– Gradually move towards gene (Fig. 8.3 P157)
Human Genome Project
Methodology - FiSH

Fluorescence in-situ hybridisation
– Use metaphase chromosomes
– Probes fluorescently labelled
– Highlight chromosome on which a specific
sequence or gene is located
– (antibody technology used allows labelling of
more than one site on the same sample )
– Use of interphase chromosomes gives 50kbp
resolution
Human Genome Project
Methodology - Linkage Studies
– Find linkages between genes
 Linkage mapping from genetic studies
 Recombination studies
 Crossover at meiosis – frequency indicates distance
between the genes
Human Genome Project
Methodology – EST maps
Human Genome Project
Methodology – EST maps

Expressed sequence tag (EST) maps
 Partial gene sequence data of a cDNA clone,
which provides a sequence from which to
generate a probe.
– Extract mRNA
– Reverse transcribe it (RNA  complementary DNA
(cDNA))
– Use cDNA sequence to probe genome
– Finds the location of expressed genes
Human Genome Project
Methodology – STS maps

Sequence tagged site (STS) maps
 STS- PCR primer based on known
sequence (randomly found)
– Can be used to link the genetic maps to the
physical map
Applications of Gene technology

Genetic testing
– Identify gene defects

Human therapeutics
– Replace defective genes with corrected sequence in
affected tissues

Useful single gene defect disorders (monogenic)
– E.g cystic fibrosis
– E.g. Duchenne muscular dystrophy
– E.g. Huntingdon’s disease

More difficult for multiple gene defect disorders (polygenic)
e.g heart disease
– Introduce antisense DNA to produce mRNA
complementary to e.g cancer causing genes and so
prevent their translation
Cystic Fibrosis

Single gene defect
 Gene encoding a chloride ion channel protein is
incorrect sequence
 Leads to reduction in secretion of water with
mucus – sticky, thick mucus produced
 Coats airways, gut
– Prone to respiratory infection, recurrent cough
– Malnutrition due to poor secretion of digestive enzymes
– Reduced life expectancy

Genetic disorder established 1946,
 Gene isolated 1989
Cystic Fibrosis

Possible treatment
– Introduce good copy of gene into airways cells
– Use aerosol technology

Delivery methods:
–
–
–
–
Aerosol
Viral vector or Liposome containing DNA
Animal trials show good reversal
Human trials less encouraging
Duchenne Muscular
Dystrophy

Defect in gene for Dystrophin (muscle
protein)
– Onset of symptoms age 2-6
– Falling, difficulty getting up from sitting/lying
– Waddling gait
– Large calf muscles (fat deposition)

X linked gene (1987)
Duchenne Muscular
Dystrophy

Treatment
– Injection of liposomes into bloodstream

Good copy of gene introduced into muscles
– Targeting/ control of tissue specific expression
– Alternative antisense technology