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

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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