BIOTECHNOLOGY – PART 2
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How do forensic scientists determine who’s blood has been left at a crime scene?
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)
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 of the same species carry the same genes, but may have
different alleles for those genes (blue eyes vs. brown eyes)
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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)
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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
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SO???
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Assume…
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We have two samples of DNA (from different individuals)
We use a restriction enzyme to cut these DNA samples
The DNA is cut up into pieces which are called restriction fragments
Since there are differences in the sequences, the restriction enzymes will not cut
in the exact same places
Therefore, different individuals will produce restriction fragments of different
lengths
This is known as restriction fragment length polymorphism (RFLP)
Example 1:
Sample 1 got cut into 2 fragments
Sample 2 didn’t get cut at all
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o20.swf::Restriction%20Fragment%20Length%20Polymorphisms
Example 2:
Both DNA samples are cut with the same restriction enzyme.
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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
RFLP ANALYSIS
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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.
GEL ELECTROPHORESIS
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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
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Therefore, smaller fragments will travel further away from the negative electrode
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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.
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Let’s re-examine our previous example
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Let’s re-examine our previous example
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Let’s consider a different example:
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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.
SOUTHERN BLOTTING SUMMARY
1. The gel is subjected to a chemical that causes the double-stranded DNA to be
denatured into single-stranded DNA
2. 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
3. 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. The nylon membrane is placed against an X-ray film
5. The probes are radioactive and cause exposure of the X-ray film
6. This pattern imprinted on the X-ray film is called an autoradiogram
7. When the film is developed, a pattern will emerge…
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MATCH THE SUSPECT
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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.
YOU ARE NOT THE FATHER!
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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.
Look at the bands that do not match the mother’s
Who does it match?
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
PCR (POLYMERASE CHAIN REACTION)
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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 nucleotides.
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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)
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The DNA polymerase of this species is known as Taq polymerase and it functions
optimally at about 70 degrees Celsius
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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
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An RNA primer must be added first
before DNA nucleotides are added to
build a complementary strand to the
template strand
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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
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
Taq polymerase – a type of DNA
polymerase – builds complementary
strands using DNA nucleotides that
have been added to the solution
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Once the RNA primers have been laid
down, DNA polymerase III will build a
complementary strand by adding DNA
nucleotides
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When the complementary strands have been built, the cycle can repeat itself over and
over again, doubling the number of copies each time.
SIMPLIFIED PROCESS…
Cool to 50 degrees
http://www.youtube.com/watch?v=eEcy9k_KsDI