Genetic fingerprintingEveryone’s DNA is unique

One way of distinguishing individuals – sequence their genome

Impractical

Alternatively, exploit differences that are unique, but easily detected

Genetic fingerprinting Within the genome are repeated core sequences (minisatellites; 12

to 100 bp, up to 3000 repeats)

The number of repeats varies – they are called variable number tandem repeats (VNTRs – equivalent to alleles for genes)

Digestion of DNA with specific restriction enzymes produces lengths of DNA (fragments). The enzymes do not cut in the minisatellites.

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 fingerprintingDetection of VNTRs

Extract 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 fingerprintingFor 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:

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

Chance of two individuals being the same? 1 in 380 = 0.003

If VNTR is located at 4 loci Chance of two individuals being the same?

(1 in 380)4 = 1 in 20,000,000,000

In practice less (fewer than 380 variants)

DNA Database 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

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 plantsProduction:

Introduce DNA Requires vector

Regenerate whole plants (clones) Needs to ensure all cells contain transgene

Introducing DNA (non grasses) Dicotolydenous plants (i.e not grasses)

Use Agrobacterium tumefaciens Causes crown gall disease in plants Contains a Ti plasmid (Ti = tumour inducing)

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 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 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

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)

AnimalsLess advanced than plants

Greater ethical concernsCurrently 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 Replace selective breeding

• 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 e.g. PPL therapeutics (Edinburgh) Sheep producing -1-antitrypsin in their milk (treats

emphysema)

Web SiteAccess Excellence web site

www.accessexcellence.org

E.g VNTR (17bp) repeated 70-450 times Chance of two individuals being the same?

1 in 380 = 0.003

If VNTR is located at 4 loci Chance of two individuals being the same?

(1 in 380)4 = 1 in 20,000,000,000

In practice less (fewer than 380 variants)