Transcript Document

BIOTECHNOLOGY
PART 2

How do forensic scientists determine whose
blood has been left at a crime scene?
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How are paternity tests conducted?
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ANSWER:
 Using restriction enzymes, a technique called Gel
Electrophoresis, and a concept called RFLP
(restriction fragment length polymorphism)
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Individuals have differences in their DNA
sequences.
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Polymorphism:
 any difference in DNA sequence (coding or non-
coding) that can be detected between individuals
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Polymorphisms can occur in both coding
regions and non-coding regions
Example 1 – polymorphisms in coding regions:
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Organisms that are the same species carry
the same genes, but may have different
alleles for those genes (blue eyes vs. brown
eyes)
Can also be used to detect mutations
(someone with sickle cell anemia will have a
different allele for one of the hemoglobin
proteins which gives the rbc a sickle shape)
Example 2 – polymorphisms in non-coding
regions:
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Non-coding regions of DNA often contain
sequences in which base pairs repeat over
and over again (these are known as variable
number tandem repeats or microsatellites)
Variations in these microsatellites exist
between individuals of the same species
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Therefore, different individuals will have
variations (polymorphisms) between their
DNA sequences
SO??
Assume …
o we have two samples of DNA (from different individuals)
o we use a restriction enzyme to cut these DNA samples
o The DNA is cut up into pieces which are called restriction
fragments
o Since there are differences in the sequences, the
restriction enzymes will not cut in the exact same
places
o Therefore, different individuals will produce
restriction fragments of different lengths
This is known as restriction fragment length
polymorphism (RFLP)
SAMPLE 1
SAMPLE 2
When separated, Sample 1 will produce a very different banding pattern
than sample 2. Hence the 2 can be differentiated.
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Both DNA samples are cut with the same restriction
enzyme

The enzyme does not cut in the exact same places
since there is variation in their DNA sequences
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These fragments can then be run on a gel (using a
technique called gel electrophoresis) which
separates the fragments according to length
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0Polymorphisms
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RFLP can be used to identify individuals.
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Scenario: A crime is committed and there are
3 suspects.
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If the criminal left a sample of blood at the
crime scene, RFLPs from this sample can be
compared to RFLPs from blood of all suspects
to determine who the criminal is.
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After restriction enzymes have been used to cut
DNA, DNA fragments must be separated and
purified from each other for analysis using a
process called Gel Electrophoresis
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In this process, the DNA fragments are placed in
wells on a gel called agarose (agar)
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An electrical current is passed through the gel
and since DNA is negatively charged, the
fragments of DNA are attracted to the positive
end.
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As the DNA fragments move through the agar,
the small fragments move quickly, but larger
fragments move slowly because they get caught
up in the gel (fibrous matrix)
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Therefore, smaller fragments will travel further
away from the negative electrode

A blue dye is added initially to the DNA
fragments. When the dyed fragments reach the
positive end, the power is turned off.
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Once the gel electrophoresis is complete, a
fluorescent dye is then used to stain the DNA
fragments.
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Ideally gel electrophoresis creates a band
pattern that is unique to each individual.
Let’s re-examine our previous example:
Which is the positive end
and which is the negative
end?
Consider a different example:
Chromosome A contains a
sequence detected by a restriction
enzyme
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In reality, the amount of DNA is usually so
large and the bands too numerous that
instead of seeing individual bands, a large
smear is seen on the gel.
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In a process called Southern blotting, the
DNA from the gel can be transferred from the
agarose to a nylon membrane.
1.
2.
3.
The gel is subjected to a chemical that causes
the double-stranded DNA to be denatured into
single-stranded DNA
Single stranded DNA is transferred to a nylon
membrane (this is the blotting). The nylon
membrane is places on the gel with a positive
electrode behind it to attract the negative DNA
The nylon is placed in a solution with
radioactive probes (complimentary
nucleotides) that will bind to specific regions of
the DNA that have been chosen by the scientist
(areas of mutation etc.)
4.
5.
6.
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The nylon membrane is placed against an Xray film
The probes are radioactive and cause
exposure of the X-ray film
This pattern imprinted on the X-ray film is
called an autoradiogram
When the film is developed, a pattern will
emerge…
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•
The band pattern of suspect
1 matches the specimen.
Thus, suspect 1 is probably
our criminal
•
The specimen must also be
compared to the victim
because the victim’s blood
may be mixed up in the
specimen or it could just be
the victim’s blood.
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In paternity test, the child’s DNA is
compared to its mother’s and the
possible fathers. Since the child has
DNA from both its mom and dad,
the bands that match its mother
can be ignored.
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Look at the bands that do not
match the mother’s
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Who does it match?
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The reason the child doesn’t have
the exact same DNA of its parents is
because it only receives half of each
parent’s chromosomes/DNA
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PCR (polymerase chain reaction) is a technique
used to clone (amplify) DNA
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Before the technique was developed, it took a
very long time to make a copy of a DNA
sequence
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A scientist would have to place the gene into a
plasmid and then wait for the bacterial cell to
make more copies during replication
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The scientist would then have to cut the plasmid
and remove the DNA sequence of interest
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With PCR, many copies of DNA can be made
quickly
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This is particularly useful when only a small
sample of original DNA is available
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(Ex: If only a small sample of DNA is obtained
from a crime scene, PCR may be used to
allow for multiple forensic tests.)
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Before running a DNA sample through the
Gel Electrophoresis for analysis, the sample
will undergo PCR to make sure there is
enough DNA for adequate testing
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The process of PCR is closely related to DNA
replication that occurs within the nucleus of a
cell.
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The PCR procedure requires primers, DNA
polymerase and DNA nucleotides.
The DNA polymerase used is from a bacteria called
Thermus aquaticus. This bacteria is a thermophile and
thrives at high temperatures (lives in hot springs)
The DNA polymerase of this species is known as Taq
polymerase and it functions optimally at about 70
degrees Celsius
Taq polymerase is used because it does not denature
at the high temperatures needed for the PCR
procedure
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DNA REPLICATION
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The 2 DNA strands are
separated using
enzymes helicase and
gyrase
PCR
The 2 strands of DNA
are separated using
heat
 At temps of 94°C – 96
°C, the hydrogen
bonds between the
complimentary strand
will break, separating
the strands
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DNA REPLICATION
•
An RNA primer must
be added first before
DNA nucleotides are
added to build a
complementary
strand to the
template strand
PCR
A DNA primer is used
instead because they are
easy to produce in labs.
• One of the primers is
known as a forward
primer and the other is a
reverse primer because
they start synthesis of
DNA in opposite
directions
• The temp must be
lowered to 50°C - 65°C in
order for the primers to
anneal to the template
DNA
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DNA REPLICATION
•
PCR
Once the RNA primers • Taq polymerase – a
have been laid down,
type of DNA
DNA polymerase III will
polymerase – builds
build a complementary
complementary
strand by adding DNA
strands using DNA
nucleotides
nucleotides that have
been added to the
solution
• When the complementary strands have been built,
the cycle can repeat itself over and over again,
doubling the number of copies each time.
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