Jan 13, 2016
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)