Transcript DNA Analysis

February 7
2014
What are the most common sources of DNA evidence?
◦ Blood and bodily fluids
◦ Tissue samples, esp skin
◦ Hair root sometimes
Investigators collect blood or buccal
swabs (inner cheek cells) for reference samples.
DNA storage
◦ Blood and bodily fluids should be frozen
◦ Tissue samples should be dried, then frozen
Before analysis, DNA must be extracted from cells.
1.
2.
3.
4.
Soaps are used to dissolve the cell and nuclear
membrane.
Enzymes are used to break down the proteins that
are bound to DNA.
Salt is used to stabilize
the DNA
Alcohol is used to
precipitate the DNA
DNA extraction from banana –
remember 9th grade?
Often investigators find very little DNA at a crime scene,
so it must be amplified, or copied, before analysis.
DNA is amplified using a process called polymerase
chain reaction (PCR).
1. Heat is used to separate the two
strands of DNA.
1
2. The solution is cooled and primers –
short pieces of nucleotides that can
begin the replication process -- are
added.
2
3. Taq polymerase, an enzyme that will
attach individual nucleotides to the
growing strands is added.
4. Once the chain is complete, the
process is repeated.
The amount of DNA doubles each cycle.
In three hours, one million copies can
be made.
Watch me!
3
4
What are the steps to processing DNA that we have
discussed so far?
1. DNA collection – collecting evidence
2. DNA extraction – extracting DNA from cells
3. PCR – amplifying (copying) the DNA
These steps would be done in any DNA forensic
investigation.
The next step is to analyze the DNA. There are
many different methods of doing this.
1. DNA samples are cut into small pieces using restriction
enzymes. Restriction enzymes recognize specific
sequences of bases and cut the DNA at that point.
Example: EcoRi recognizes the sequence GAATTC
and cuts between the G and the A.
GA A T T C
CT T A A G
Restriction
Fragment
Length
Polymorphism
This cuts the DNA into fragments of different
lengths. Different people will have different lengths
of fragments, based on the sequence of their DNA.
1. DNA samples are cut into small pieces using restriction
enzymes. Restriction enzymes recognize specific sequences
of bases and cut the DNA at that point.
2. Next, the fragments are sorted by size using gel
electrophoresis.
How does gel electrophoresis separate fragments by
size?
DNA is negatively charged, so it moves through the
gel towards the positive electrode. Small pieces diffuse
faster than large pieces, so the fragments end up forming
distinct bands according to length, with small ones closer
to the + end and large ones closer to the sample wells.
1. DNA samples are cut into small pieces using restriction
enzymes. Restriction enzymes recognize specific
sequences of bases and cut the DNA at that point.
2. Next, the fragments are sorted by size using gel
electrophoresis.
3. DNA fragments are visualized in some way (such as
radioactive probes).
4. The number and location of different-sized fragments
of DNA can then be analyzed.
Watch me!
Shown here are
fragments of
DNA that have
been cut with
EcoRi, then
separated
according to
length with
electrophoresis.
Where is the positive end of the gel and
where is the negative, and how can you
tell?
Which fragment is which?
Shown here are
strands of DNA from
two different
people.
The restriction sites
for a particular
enzyme are marked
with horizontal
lines.

Work in groups of 2-3

Get a sheet of yellow lined paper.

Cut it into four pieces length-wise.


Tape the pieces together so that you have one long
strip of paper.
Write bases (A, C, G, T), one on each line down the
length of the paper.

Add the complementary bases next to them.

This is your DNA strand.

You will now cut your piece of DNA into pieces
using the simulated restriction enzyme TWI. This
enzyme recognizes the sequence “A T” and cuts
between the A and the T.
Example:
Two
restriction
sites
A
C
C
T
A
C
A
T
G
C
A
T
G
T
G
G
A
T
G
T
A
G
G
T
A
C
Note that two
strands are
read in
opposite
direction.
This is
because DNA
is antiparallel.



You will now simulate
electrophoresis by sorting
your strands of DNA
according to size.
Count how many lines
long each piece of DNA is.
We will make a class ‘gel’
on the board. Pick one
lane and shade in the
boxes that represent the
lengths of your fragments.
Size
70-104
65-70
55-65
50-55
45-50
40-45
35-40
25-30
20-25
15-20
10-15
5-10
0-5
Group 1 Group 2