biotechnology 4 major areas human genome project gene therapy forensic science agriculture
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BiotechnologyBiotechnology
4 major areas
Human Genome ProjectGene TherapyForensic scienceAgriculture
Human Genome ProjectHuman 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 – Human Genome Project – Approach UsedApproach Used
Concept– Produce ever more detailed maps of chromosomes– 1. Genetic linkage map (low resolution)
Relative order & spacing of disease linked genes (not physical map)
– 2. Combine with STS/EST (sequence tag site/ expressed sequence tag) maps
Position of unique DNA sequences (physical map) Linkage data to disease genes
– Pain stakingly slow, but links to useful disease information– Alternatively SHOTGUN sequencing
–3. Chop chromosome - small, overlapping fragments–Sequence–Computers align overlapping sequences
Human Genome ProjectHuman Genome Project
Methods
Restriction Enzymes - Restriction Enzymes - SummarySummary
Variety of enzymes Isolated from bacteria
Cut DNA at specific sequencesUsed to produce DNA fragments
– Blunt or sticky endedDNA Ligase (not a RE), used to LIGATE
(joins DNA) fragment into a plasmidAnimation
Pst1
BamHI
BamHI
BamHI
11kb
3kb
6kb
BamHI
PvuI
PvuI
HindIII
1236bp
1875 bp
900bp
1670bp
DNA AmplificationDNA 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?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 Electrophoresis Gel PreparationPreparation
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!Comb
Tray
Molten agarose 55 - 60°C
DNA SIZE MARKERS/STANDARDSDNA SIZE MARKERS/STANDARDS
Smaller fragments travel faster.The sizes of bands are known
(in base pairs).largest
smallest
-ve
+ve
Typical DNA gel showing bands of
DNA of different sizes.
First and last lanes contain DNA size markers
DNA sequencingDNA 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 probesDNA 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 Using a Probe to Find Sequences on a GelSequences on a Gel
Usually a nitrocellulose membrane
DNA on the gelis double stranded & needs to be single-stranded for probe to bind: gel treatedwith sodium hydroxideto do this
Chromosome WalkingChromosome 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 Human Genome Project Methodology - FiSHMethodology - 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 Human Genome Project Methodology - Linkage StudiesMethodology - 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 Human Genome Project Methodology – EST mapsMethodology – EST maps
Human Genome Project Human Genome Project Methodology – EST mapsMethodology – 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 Human Genome Project Methodology – STS mapsMethodology – STS maps
Sequence tagged site (STS) mapsSTS- PCR primer based on known
sequence (randomly found) – Can be used to link the genetic maps to the
physical map
Applications of Gene technologyApplications 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 FibrosisCystic 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 FibrosisCystic 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 Duchenne Muscular DystrophyDystrophy
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 Duchenne Muscular DystrophyDystrophy
Treatment– Injection of liposomes into bloodstream
Good copy of gene introduced into muscles– Targeting/ control of tissue specific expression– Alternative antisense technology
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