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
–3. Chop linkage
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
–Computers
Position
alignof unique DNA sequences (physical map)
Linkage
overlapping data to disease genes
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
During polymerisation
Molten agarose 55 - 60°C
the sugar molecules
all cross link with
each other causing
the solution to ‘gel’
into a semi-solid
matrix; a bit like jelly
Comb in a trifle!
Tray
DNA SIZE MARKERS/STANDARDS
-ve
Smaller fragments travel faster.
largest The sizes of bands are known
(in base pairs).
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 single-
stranded 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
