Download CSI: SNAB - NKS | VLE - our Online Classroom

CSI: SNAB
DNA Fingerprinting
Lesson Objectives
Describe the uses for DNA Fingerprinting
Explain the process of DNA Fingerprinting
Discuss the ethics behind DNA
Figerprinting
What is DNA Fingerprinting?
DNA Fingerprinting (or DNA Profiling) is a
technique using DNA to create a unique
pattern of DNA fragments to identify an
individual
DNA fingerprinting is not DNA sequencing
and the exact sequence of the DNA is not
discovered
Applications of DNA Fingerprinting
DNA fingerprinting has a variety of uses:
Matching unknown biological samples to
known samples. This is used in:
– Forensic Science
– Pet Recovery services
Identifying a family relationship
(mother/father/siblings etc)
Identifying a species e.g. Tiger DNA from
a pelt, useful in conservation
Forensic DNA Fingerprinting
This technique can be used to match a
biological specimen to an individual/ suspect
Blood
Skin (Epithelial Cells)
Semen
Saliva (as it often contained Epithelial Cells)
Hair (From cells in the Root Sac)
Any biological sample which contains cells!
Steps to DNA Fingerprinting
Isolate a sample of DNA
Use Polymerase Chain Reaction to
amplify DNA
Treat DNA with Restriction Enzymes
Separate DNA Gel Electrophoresis
Probe DNA using Southern Blotting
Interpretation
Why are People’s DNA different
The difference between the coding DNA
(that’s the DNA in genes) is very small!
There is however a great difference in the
non-coding DNA between genes and in
Introns (non coding DNA within a gene)
Mini and Micro Satellites
In the non coding DNA (introns) there
are short sequences of DNA which are
repeated many times. These are known
as Satellites
Mini-satellites contain 20-50 base pairs
and are repeated 50 to several hundred
times
Micro-satellites contain 2-4 base pairs
and are repeated between 5 and 10 times
Satellites and Loci
Satellites occur at particular places in the
human genome
These places are called Loci (or a Locus)
The number of repeats at each locus
varies from person to person and even
vary between a person’s two
chromosomes (Maternal and Paternal)
This variation is caused by the process of
Crossing Over during meiosis
Example of Satellites at a Locus
Mini-satellite repeat (VNTR – Variable Number Tandem Repeat)
Polymerase Chain Reaction
PCR is a very modern technique designed
to amplify (make large numbers of copies)
a small amount of DNA
PCR requires the following:
– A DNA Sample
– DNA Primers
– DNA Polymerase
– Solution of free nucleotides
DNA Primers and Polymerase
DNA Primers are short sequences of DNA which
bind to the the sequence at the start of a microsatellite repeat
The enzyme DNA Polymerase then copies the
section of DNA at the locus using the free
nucleotides in solution
This DNA polymerase is very special as it has
been obtained from thermophillus (heat loving)
bacteria which works even at 95 degrees!
In the UK ten primers are used to amplify ten loci
Hot water bacteria:
Thermus aquaticus
Taq DNA polymerase
Life at High Temperatures
by Thomas D. Brock
Biotechnology in Yellowstone
© 1994 Yellowstone Association for Natural Science
http://www.bact.wisc.edu/Bact303/b27
Restriction Enzymes
The DNA sample is treated with
Restriction Enzymes
These Enzymes cut DNA at specific sites
dictated by the sequence at that site
The DNA is cut up into a series of lengths
These lengths vary in size between
individuals (even animals!)
DNA Restriction Enzyme Site
Restriction Enzymes are Enzymes That Cut DNA Only at
Particular Sequences
The enzyme EcoRI cutting DNA at its recognition sequence
Different restriction enzymes have different recognition sequences.
Gel Electrophoresis
Gel Electrophoresis is a technique used to
separate molecules by their size (and
even by charge)
It can be used to separate different sizes
of lengths of DNA
How it works
A gel is made from a chemical called agarose
(made from seaweed!)
Samples of unknown DNA and DNA of known
lengths are loaded at one end of the gel
An electrical current is passed along the gel
As DNA is mainly negative it is attracted to the
positive electrode
The smaller the DNA fragment the quicker and
further it travels along the gel
Making the gel
Adding samples
Electrophoresis
Southern Blotting
The fragments of DNA within the gel are
transferred to a nylon membrane and washed
over with a DNA probe that binds to the
repeated sequence
The membrane is then placed in a bag and
placed on a photographic film which is exposed
where the radioactive probes are attached
The resulting pattern of bands is called the DNA
fingerprint
A single band occurs where the maternal and
paternal chromosomes have the same number
of satellite repeats, if not there will be two bands
Summary
Automation
This shows the result of a modern automated
technique using fluorescent DNA to make a
computer generated graph
Amelogenin
The genes for amelogenin can be used in sex determination of
samples from unknown human origin through the Polymerase Chain
Reaction (PCR).
Using primers specific for intron 1 of the gene, the gene sequence
for the intron can be amplified. The X chromosome gene, AMELX,
gives rise to a 106 bp amplification product (amplicon) and the Y
chromosome gene, AMELY, a 112 bp amplicon. Hence, the AMELX
contains a 6 bp deletion in the intron 1.
When the amplicons are run on an agarose gel, samples from male
sources (XY) will show two bands on an agarose gel (one for the
106 bp fragment and one for the 112 bp fragment), while females
(XX) will show only one band.
Thus, this process allows for sex determination of unknown
samples.
Interpretation
In the UK ten loci are copied during DNA
fingerprinting
In the USA thirteen loci (they call them
alleles) are copied, why?
Because the USA has a larger population
and so there is more chance of a
similar/identical DNA fingerprint
Even so this is still very unlikely
Matching Loci
To match a sample of DNA all ten loci
must be of the same length!
If even one is different there is no match
The defendant stated that the blood on his clothing was his.
The First Case
22/11/83 - Lynda Mann was found
murdered
No clues but the murderer/rapist had left a
semen sample.
Four years later - 31/7/87 - Dawn Ashcroft
was found murdered and raped and there
were enough similarities between the
cases for the police to link them.
The First Case
A massive man hunt was launched
resulting in the arrest of a young
dishwasher.
He confessed to the murder of Dawn but
would not admit to the murder of Lynne.
DNA from the suspect was compared to
that from the samples found at both crime
scenes.
The results were surprising.
The First Case
The suspects DNA did not match either of
the samples and therefore the dishwasher
was innocent of both murders.
As a result a huge DNA screening of the
local population was undertaken without
success.
The killer and rapist was eventually
identified as Colin Pitchfork after a tip-off
from a work colleague.
DNA and Paternity
The DNA fingerprints of children share loci
with both parents
DNA and paternity testing
A
B
C
D
E
A mother
B male 1
C male 2
D child
E standards
DNA and paternity testing
Which is the father of child D?
Male 1 because the child has one allele
matching male 1 and one matching the
mother
Problems with DNA Fingerprinting
Statistics
Population genetics
Technical difficulties
DNA Databases
In England and Wales, anyone arrested on suspicion of a recordable
offence must submit a DNA sample to the database, which is then
kept on permanent record.
In Scotland, the law is different and most people are removed from
the database if they are acquitted. In Sweden, only criminals who
have spent more than two years in prison are recorded.
In Norway and Germany, court orders are required, and are only
available, respectively, for serious offenders and for those convicted
of certain offences and likely to reoffend.
All 50 states in the USA keep profiles of violent offenders, and a few
keep profiles of suspects.
Portugal has plans to introduce a DNA database of its entire
population