Applications of DNA Technology
DNA technology, forefront, cell biology, potential, solve
problems known, future potential, discovered.
Main areas studied:
•Human Genome project
•Human Therapeutics
•Forensic Science
•Agriculture
Human Genome Project
15 year project (mainly USA, Europe and Japan)
Map genome
Isolate genes
Determine function of genes
Compare to other species maps
Explore ethical, social, legal issues
Progress
2004 genome mapped, nucleotide sequence for
every human chromosome known.
3 billion nucleotide base pairs in human DNA
1 - 2% (20 - 40 thousand genes) of the 3 billion is
essential for protein coding sequence (extrons).
98% regulation of gene transcription or unknown
function (such as spacers between genes or in genes
(introns))
DNA analysis, complicated, time consuming,
greatly enhanced by new technology especially
computers.
3 main methods for DNA analysis
Genetic (linkage) mapping
Physical mapping
DNA sequencing
Genetic linkage mapping
Locates relative position of genes on chromosomes,
cross over values (COV) or recombination frequencies in
meiosis.
•Used for inherited characteristics
•Used for gene mutations that cause genetic disorders
•use short tandem repeats (micro-satellites) of repeating
base sequences e.g. CACACACACA of variable length
Physical mapping
Increased resolution (detail) needed.
Starting with long pieces of DNA and working down.
This needed new technology and techniques including:
•Cutting DNA up using restriction endonuclease enzymes
•Cloning DNA using micro-organisms (bacteria)
•Identifying genes or micro-satellites using nucleic acid probes
•Use chromosome walking to sequence genetic markers
Restriction mapping
DNA fragments are generated using restriction endonucleases
enzymes.
Enzymes recognise specific short sequences of base pairs usually
(4 - 8 nucleotides long). Different enzyme different sequence
(100 or so enzymes from bacteria)
Fragments are identified by size or by attaching a flourescent tag
(were radioactive)
When put back with unzipped DNA strand these short tagged
sections would reveal location of sequence of nucleotides
(chromosomal walking).
Many repeats with different lengths of DNA gave overlapping
picture of sequence of nucleotides
Polymerase Chain Reaction (PCR)
Advances in producing cloned DNA
Alternative method of producing more DNA is the
polymerase chain reaction (PCR).
No living cells are needed, machines produce in hours
what micro-organisms would have done in days.
Very useful where small quantities of DNA are available
such as samples of hair with follicle, saliva, blood or
semen.
Using PCR to clone DNA
•provide DNA to be copied,
•four bases (as deoxyribose nucleotide phosphates)
•DNA Taq polymerase enzyme (heat stable),
•two primers (short lengths of single strand synthetic
DNA complementary to the 3' end to be copied)
Using PCR to clone DNA
o
•Separate strands of DNA by heating to 95 C for 30 seconds.
o
•Add primers and cool to 55 C for 20 seconds so primers can
anneal (join) at correct sequence.
o
•Heat to 75 C to allow DNA Taq polymerase enzyme to
join nucleotides to corresponding bases.
•Allow sequence to repeat itself automatically to produce
many clones of original DNA.
•Fully automated process
DNA sequencing
Once gene position had been worked out, base pair sequence was
then determined using a process similar to PCR and gel
electrophoresis.
A machine capable of building DNA sequences like PCR is used but
altered tagged nucleotides are used that can halt the replication
process.
The process is allowed to run for increasing lengths of time giving
many different lengths of DNA. These are then separated out
using gel electrophoresis sensitive enough to separate fragments
differing by only 1 nucleotide.
DNA sequencing
Technology now allows sequencing of DNA
fragments very quickly.
Computer hardware and software much more
sophisticated and improving.
For example gel electrophoresis is now obsolete
and other techniques are used.
Genome studies
Put simply the size of organism relates to size of genome,
but this is not always the case there are exceptions.
Reasearch done on mainly bacterium (E.coli), yeast
(S.cerevisiae), nematode worm (C.elegans), fruit fly
(D.melanogaster) and mouse (M.musculus).
There are many shared genes and DNA sequences between
organisms.
Humans and chimpanzees 98% identical DNA sequences.
Genome Sizes
Organism
genome size
number of
(Mb)
genes
E.coli
4.6
4,405
S.cerevisiae
12.1
5,800
D.melanogaster
150
12,200
H.sapiens
3,000
30,000
N.tabacun
4,500
43,000
Human Therapuetics
Congenital abnormalities (genetically based diseases)
Single gene defects (monogenic trait)
5,000 described and characterised as either:
•autosomal dominant
•autosomal recessive
•X linked (most are recessive if X linked)
Multiple gene defects (polygenic trait)
Detecting Genetic Disorders
Disease symptoms and historical information
Family relationships/pedigree analysis to determine nature
(e.g.dominant/recessive/x linked)
Monogenic trait identified with gene analysis techniques:
gene mapping, physical mapping, DNA sequencing
used to determine location and sequence of base and its effects.
Treatments can then be derived and genetic counselling given
Cystic fibrosis and Duchennes muscular dystrophy
Cystic Fibrosis
Autosomal recessive monogenic trait (affecting 1 in 2,000)
Gene of membrane carrier protein of 1,480 aa (complex
structure including 2 transmembrane domains, 2 ATP binding
domains and a regulatory region).
Mutations in gene give defective ion transport system
(epithelial cells not fully hydrated), sticky mucus in the lungs.
Major symptoms include inflammation of lung tissue and
persistant bacterial infections
Other problems include defects in pancreatic function,
infertility and increased risk of diabetes.
Diagnosis - abnormally salty sweat.
Quality of life reduced/life expectancy 30 years.
Defective CF gene carrier frequency of 1 in 22.
In parents where both carry defect 1 in 4 chance of any child
receiving both recessive alleles.
Mapped to chromosome 7.
550 mutations described so far.
Most common is deletion of 3 base pairs removing 1 amino acid
((F508) Deletion, phenylalanine, 508th amino acid)
Defective protein does not fold properly and does not reach
membrane location.
Duchennes Muscular Dystrophy
•X linked affecting 1 in 3,300 boys.
•progressive wasting of muscles, confinement to wheel chair
in teenage years.
•Wasting, paralysis, respiratory difficulties.
•life expectancy 30 years (approx).
•gene found in 1987
•protein dystrophin 3,685 aa. Normal function is to link
cytoskeleton with muscle cell membrane (sarcolemma) in
muscle cells.
Screening tests
When?, Why?, How?
•best done before conception
•voluntary but only usually done on families with a
history of either
•25% chance of having an affected child if both are
carriers
•prenatal genetic testing (amniocentesis or chorionic
villus (placenta) sampling), only if parents willing to
have an abortion
•postnatal tests: excessively salty sweat for CF and
raised creatine kinase levels for DMD.
Uses of Gene therapy
Use of genes to treat illnesses by:
•killer genes to target tumours so that only the cells
with the genes are affected by drug treatment.
•Replacing defective genes in cells
•Inserting extra genes to have a beneficial effect
Use of Gene Therapy
Use of gene therapy very complex:
•normal gene must be available in cloned form
•affected cells must be accessible either in vivo
(patient) or in vitro (tissue culture) then transplanted
•suitable vehicle (vector) for delivery of gene
•gene functions normally in target cells
Use of Gene Therapy
Vectors for delivery include:
•Liposomes (hollow membrane sphere surrounding a
plasmid). The plasmid delivers the DNA to the target cells.
Problems are that plasmids do not incorporate themselves
into the chromosomes so short term expression.
•Virus (recombinant virus) replication genes are removed and
space filled with therapuetic gene.
Virus can then integrate its genetic material with host cell.
Problems of immune response by host organism and specific
host cells invaded by virus particles
Gene Therapy
Ethics
•somatic cells?
•germ cells?
•Counselling
testing for defects
treatment of defects
Forensic uses for DNA Technology
Criminal
Paternity
Immigration
Criminal
DNA 'fingerprinting' developed in 1984
Uses fragment size of DNA molecules to give a banding pattern
from gel electrophoresis.
Process is:
•Isolate DNA
•Digest with enzymes
•Transfer DNA fragments to filter
•Bind a labelled probe of DNA to known locations, run gel
electrophoresis
•Analyse banding
•Single locus probes
Alec Jeffreys
DNA banding patterns
Probe
•single locus probes
•bind to 1 complimentary sequence in genome.
•2 bands are visible in autoradiogram (paternal and
maternal).
DNA profiling
DNA profiling now used more often than DNA fingerprinting.
Process is:
isolate DNA sample
amplify (clone) using PCR but use a primer that will bind to a
specific short tandem repeat
repeat with 3 or 4 different DNA primers
label the cloned DNA with fluorescent dyes.
Analyse using automated DNA sequencer to produce a chart
with peaks showing length of each cloned fragment
look for matching peaks in samples analysed
DNA profiling
Reliability of Techniques
Quality control:
•recording of samples
•cross checking of procedures
•contamination by other DNA during PCR
•Inspection of procedures
Public confidence
Legal fortitude
Agriculture
Great potential
Applied to both animals and plants
Genetic modification to improve species which is benefit to
producer and consumer
Plants
resistance to disease, drought, herbicides,
pesticides
increasing yield/growth, fruit shelf life,
novel products - oils/plastics, vaccines, antibodies
Animals increasing yield/growth, organs for transplant
Problems acceptance by public
consequences of genetic modification
Genetic Modification
TRANSGENIC organisms
genes for the desired characteristic from one
organism inserted into the hosts DNA.
Genetic material to future generations.
(Revise Recombinant DNA technology to produce
Insulin from E.coli bacteria)
Transgenic plants
Requirements
vector to insert cloned DNA fragment into plant cell
genome
method of regenerating whole plants so all cells
carry the transgene.
Tissue culture in plants
1 vector is Agrobacterium tumefaciens (soil bacteria).
Can insert DNA from a plasmid into plant cells
Ti plasmid used (usually induces crown gall disease in plants)
(Ti = tumour inducing).
Ti plasmid carries T-DNA a region that integrates with plant cell
genome.
Ti plasmid reduced in size and target gene incorporated into TDNA region.
The smaller plasmids lack the tumour producing effects.
Producing a transgenic plant
Transgenic plant examples
Tomato plants - delayed ripening by inhibiting the production
of ethylene (normally produces ripening), fruit stays firmer
longer.
Biotech company CALGENE produce Flavr Savr tomato.
Insect resistance by inserting gene for insecticidal protein.
Nitrogen fixation in non legumes
Cereals/grasses unfortunately are not affected by Ti plasmid,
so new insertion methods are needed.
Problems with transgenic plants
Monsanto produced soya beans resistant to round up
weed killer.
So what?
Transgenic Animals
Transgenic animals include:
sheep that can make antitrypsin in milk to treat emphysema.
Another important use of DNA Technology and animals
are products that are given to animals including
hormones, antibodies, vaccines, cellulase produced by
genetic engineering
Bovine growth hormone as an example
Bovine somatotrophin (BST) produced by Monsanto
Grown using bacteria and injected into cattle in America
Increases milk yield by 10%
Should not effect any one who eats meat.
Public concern over containment of the hormone so
importation of meat or from cattle treated with this is
banned
Producing
bST to give
to cows.
Future Questions
Transgenic crops good/bad?
Information?
Choices?
Manipulation by big companies?
Ensuring food supplies?
Future?
vaccine production
disease diagnosis
pharmaceutical proteins
xenotransplantation (organs grown in pigs)