Biotech Continued….
4.4

How do forensic scientists determine
who’s blood has been left at a crime
scene?

How are paternity tests conducted?

ANSWER: Using restriction enzymes, a
technique called Gel Electrophoresis, and
a concept called RFLP (restriction
fragment length polymorphism)

Individuals have differences in their DNA
sequences.

Differences in coding regions (exons) may
result in a mutation (ie. Sickle cell anemia)

Differences in noncoding regions (introns)
may exist in the form of variations in the
number of repeating units
RFLP analysis

If a sample of DNA is digested by a restriction
enzyme, the result would be the formation of a
number of DNA fragments of various lengths.

Since we all have different DNA (because we all
have different numbers of VNTR) exposure to
the same restriction enzyme would produce
different numbers and lengths of fragments.

This is known as RESTRICTION FRAGMENTS
LENGTH POLYMORPHISM

RFLP can be used to identify individuals.

Scenario: A crime is committed and there
are 3 suspects.

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





The DNA fragments must be separated and
purified from each other for analysis using a
process called gel electrophoresis
In this process, the DNA fragments are placed in
wells on a “jello-like gel- called agarose (or
agar)
The agarose is fibrous and acts like a net
An electrical current is passed through the gel
Since DNA is negatively charged, the fragments
of DNA are attracted to the positive end.

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 fibres of the gel.

A blue dye is added initially to the DNA
fragments. When the dye reaches the
positive end, the power is turned off.

A fluorescent dye is then used to stain the
DNA fragments.

Ideally this creates a band pattern that is
unique to each individual.
Molecular Markers are
DNA fragments of
known lengths. They
may be run during a
gel electrophoresis so
that the fragments of
the sample DNA can
be compared to the
markers to determine
approximate length.

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.

In a process called SOUTHERN BLOTTING, the
DNA from the gel can be transferred to a nylon
membrane.

The nylon membrane is placed against an X-ray
film to read an autoradiogram which will show
the band pattern
Match the Suspect

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.
Who’s your Daddy???

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

PCR is a technique used to clone (amplify)
DNA

Before the technique was developed, if
scientists wanted to make multiple copies
of a gene fragment, the gene would have
to be inserted into a plasmid and the
bacterial cell would make more copies
when it replicates its plasmids. Then
scientists would have to remove the
plasmids and cut out the bacterial genes.

With PCR, many copies of DNA can be
made quickly.

This is particularly useful when only a
small sample of original DNA is available.

(Ex: If only a small sample of DNA is
obtained from a crime scene, PCR may be
used to allow for multiple forensic tests.)

Before running a DNA through the Gel
Electrophoresis for analysis, the sample
will undergo PCR to make sure there is
enough DNA for adequate testing.

The process of PCR is closely related to
DNA replication that occurs within the
nucleus of a cell.
DNA REPLICATION

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

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.

(The temp is lowered to
5O°C - 65 °C to allow for the
DNA primers to attach)

One of the primers is known
as a forward primer and the
other is a reverse primer
cause they start synthesis of
DNA in opposite directions
DNA REPLICATION

PCR
Once the RNA primers  Temp raised to 76°C
have been laid down,  Taq polymerase – a
DNA polymerase III
type of DNA
will build a
polymerase – builds
complementary
complementary
strand by adding DNA
strands using DNA
nucleotides
nucleotides that have
been added to the
solution
@ 76°C – the Taq polymerase
does not denature because it is
extracted from a bacteria that
lives in hot springs (Used to hot
temps!)

When the complementary strands have been
build, the cycle can repeat itself over and over
again, doubling the number of copies each time.
Simplified process….
Animations

http://highered.mcgrawhill.com/sites/0072437316/student_view0/
chapter16/animations.html#