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Genetic fingerprinting
 Everyone’s
DNA is unique
One way of distinguishing individuals –
sequence their genome
 Impractical
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 Alternatively,
exploit differences that are
unique, but easily detected
Genetic fingerprinting
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Within the genome are repeated core sequences
(minisatellites; 12 to 100 bp, up to 3000 repeats)
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The number of repeats varies – they are called variable
number tandem repeats (VNTRs – equivalent to alleles for
genes)
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Digestion of DNA with specific restriction enzymes
produces lengths of DNA (fragments). The enzymes do not
cut in the minisatellites.
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Size of fragments containing VNTRs will vary between
individuals due to variation in the number of times the
sequence is repeated (Restriction fragment length
polymorphisms – RFLPs)
Genetic fingerprinting
 Detection
 Extract
of VNTRs
DNA
 Digest DNA using restriction enzyme
 Separate fragments by gel electrophoresis
 Southern blot (transfer DNA onto nylon
membrane)
 Hybridise with labelled probe which
recognises the particular repeated
sequence
Genetic Fingerprinting

VNTRs can occur only once in the genome
(single locus) or can occur in a number of
places in the genome (multilocus).
 Single locus probes are fine for paternity
cases (each individual has two VNTR
“alleles” – one from mother/ father)
 Analysis with a single locus probe will
indicate if baby has one of father’s alleles
Genetic fingerprinting
 For
criminal investigation VNTRs
located at a variety of loci are used
 Initially
6 loci used, now 14 loci
 Consequently a complex series of bands is
produced reflecting a variety of RFLPs
 Statistically identification on the order of
one in 100 million.
 Cross checking can be done using different
VNTRs
Animation
DNA Profiling

PCR based technqiue
 Simple tandem repeats (STRs)
 Similar to VNTRs, but shorter sequences are
repeated so can be “PCRed”
 Advantages:
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Automated analysis using the laser detector on a
DNA sequencer to indicate lengths of STRs
Smaller sequences less sensitive to degradation
PCR means exceptionally small amounts of DNA
can give a result (e.g DNA left by touching an
object)
Colour labelling of probes means multiple probes
can be used simultaneously speeding up the
process greatly whilst maintaining certainty
Probability

E.g VNTR (17bp) repeated 70-450 times
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Chance of two individuals being the same?
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If VNTR is located at 4 loci
Chance of two individuals being the same?
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1 in 380 = 0.003
(1 in 380)4 = 1 in 20,000,000,000
In practice less (fewer than 380 variants)
DNA Database
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Established 1995: 700,000 , by April 2000; by July, 2005
2,900,000 (~ 5%) profiles held on the database in UK (
5,000,000 by 2010)
630,000 matches made between crime scene and
suspect
40,000 leads as a result of profile
50% of UK crime scenes now yield DNA on NDNAD
Family relationships can also be detected ( and have
been)
Computer analysis now allows mixed DNA samples to be
analysed
Proposed that eye colour, hair colour of suspects can be
determined from DNA, surname?
52% of innocent DNA is from black people; 77% young
black men are on NDNAD
Transplants
Agriculture
&
Biotechnology
Gene modification/ insertion to improve
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Crop plants
 Yield
 Disease/ pest resistance
 Herbicide resistance
 Crop properties
 Vitamins
 Shelf life
 Medically useful products
 Industrially useful products (biodegradable plastics)
Animal
 Faster growth rate
 Higher yield
 Medically useful products
Quicker results than with selective breeding
 Introduce foreign species DNA
Transgenic plants
 Production:
 Introduce
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DNA
Requires vector
 Regenerate

whole plants (clones)
Needs to ensure all cells contain transgene
Introducing DNA (non
grasses)
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Dicotolydenous plants (i.e not grasses)
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Use Agrobacterium tumefaciens
Causes crown gall disease in plants
Contains a Ti plasmid (Ti = tumour inducing)
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Use a modified Ti plasmid which does not produce
tumour
or
The Ti plasmid contains a region T-DNA that integrates
into plant genome
T-DNA can be used by itself to carry useful genes
into a plant’s genome without causing tumours
Technique

Use restriction enzymes to excise T-DNA
 Insert gene of interest (sticky ends, ligase)
 Transform plant cells in tissue culture
 Grow calluses
 Manipulate hormones to grow fully functional
plants (clone more using conventional
methods)
Flavr Savr tomato
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Ripening of tomatoes is caused by enzyme
polygalacturonase
 Which breaks down the cell wall
Overripe tomatoes are more easily damaged
and don’t sell well.
An antisense copy of PG was introduced into
the tomato
It prevents production of PG (the two mRNAs
base pair and cancel each other out)
No PG, no rotting
Other examples
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Monsanto Roundup resistant soybean
 Can apply large amounts of herbicide
 Improves productivity of crop
Pest resistance genes
 Bacillus thuringiensis produces a protein,
toxic to insects
 Gene for protein inserted into tomato
Future
 Nitrogen fixation into non-leguminous plants
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Difficult as most useful crop species are
monoctoyledonous (grasses), so Ti plasmid can’t be
used
Alternatively, use DNA gun (gold, DNA coated pellets
shot directly into cells)
 Arabidopsis thaliana (thale cress) R genes
confer pesticide resistance
Possible to insert them into crop species
Stress (heat/ drought) tolerant genes
Modification of structure to improve harvesting
Nutritional improvement (added protein/ amino
acids/vitamins)
Manufacture of biodegradable plastics (monomer
polyhydroxybutyrate)
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Animals
 Less
advanced than plants
 Greater
 Currently
ethical concerns
use of biotech produced
growth hormone (Bovine somatotrophin
– BST) in cows to improve milk yield
(USA)
 Produced by transformed E.Coli.
Containing BST gene
Future
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Replace selective breeding
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Directly introduce desirable genes into animals
Tried in pigs – multiple copies of GH
Increase growth rate and ultimate size
Pigs collapsed under their own weight
Introduce genes for pharmaceutically useful
proteins into animals
Vaccines/ antibodies/ organ production
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e.g. PPL therapeutics (Edinburgh)
Sheep producing -1-antitrypsin in their milk (treats
emphysema)
Web Site
 Access
Excellence web site
 www.accessexcellence.org
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E.g VNTR (17bp) repeated 70-450 times

Chance of two individuals being the same?

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
If VNTR is located at 4 loci
Chance of two individuals being the same?


1 in 380 = 0.003
(1 in 380)4 = 1 in 20,000,000,000
In practice less (fewer than 380 variants)