Name:
CSI - Forensic Investigation
DNA fingerprints may be used to help determine guilt or innocence, paternity, the
evolutionary relationships between organisms, and for isolating genes from a genome. Our
focus in this activity will be a Crime Scene Investigation.
DNA can be acquired from any source containing human cells (blood, saliva, semen, hair,
skin….). The lab we are about to do is a simulation of the creation and analysis of a DNA
fingerprint. Your job is to become familiar with the steps associated with making a DNA
fingerprint and be able to describe the function of the tools used.
In most crime scenes the source of human cells is
very limited (drop of blood, a few body hairs, skin
under the finger nails of the victim….). In order to
create a DNA fingerprint a large amount of DNA
is needed. It becomes necessary to take the small
amount of DNA collected and make millions of
copies of it. This requires the use of a special
device called a PCR machine (aka: thermal
cycler). The collected DNA is placed in small
reaction vessels along with all four types of DNA
nucleotides and a DNA polymerase called "Taq
polymerase".
Questions:
1- Why is the PCR machine also known as a thermal cycler?
2- Why is Taq polymerase needed to support the PCR reactions?
3- What happens to the amount of DNA in a PCR machine each time it completes one cycle?
Once a sufficient amount of DNA copies has
been created it is now possible to make a
DNA fingerprint. For comparison purposes
the CSI team will usually copy and then
analyse multiple sources of DNA, such as:



DNA taken from the crime scene
victims DNA
DNA from all suspects
After copying large amounts of DNA using the
PCR machine, the next step is to mix one or
more restriction enzymes with the DNA.
Adapted by GJL from Amador Valley High School, with thanks to Mr Thiel
Name:
Restriction enzymes (aka: restriction endonucleases) can bind to DNA molecules wherever a
specific sequence of nucleotide bases exist on the DNA strand. Once locating the site where
the sequence exists, the enzyme cuts through the DNA. Some restriction enzymes cut straight
through and create blunt end fragments while others cut through leaving exposed bases. This
type of cut is referred to as a “sticky end” cut. It is called this because another DNA fragment
having the complimentary sequence of exposed bases can attach to the end of the DNA.
“Sticky end” cutting enzymes are what are used when attempting to splice a DNA fragment of
one organism into the genome of another.
Restriction enzymes were discovered in bacteria cells. It
appears that bacteria use these enzymes as a type of
immune system. Any foreign DNA that enters a bacteria cell,
from a virus perhaps, if the same sequence of bases is
present on the foreign DNA as can be recognized by the
enzyme, then the foreign DNA will be cut into pieces and
rendered harmless. Many restriction enzymes have been
discovered. A few are shown in the image on the left. Their
name is derived from the species of bacteria in which the
enzyme was first discovered.
Restriction enzymes are added to the copied DNA so that it
may be cut at multiple places and as a consequence produce
many DNA fragments of varying lengths. These are called
restriction fragment length polymorphisms or simply
“RFLP’s”. Once placed in a gel of a gel electrophoresis chamber the fragments will be spaced
out as a result of their varying lengths (largest move the shortest distance and smallest move
the greatest distance). DNA from the same source should have the same separation pattern.
Questions:
4- In what species of bacteria was the restriction enzyme EcoR1 discovered?
5- Explain why "Sticky End" cutting enzymes are necessary for supporting the movement of
DNA fragments into the genomes of other organisms.
6- Look at the base sequence on the DNA molecule below. How many places would the
restriction enzyme EcoR1 cut this piece of DNA? How many nucleotides long is each DNA
fragment?
7- How many DNA fragments would be produced if you mixed the DNA molecule above with
HindIII?
Adapted by GJL from Amador Valley High School, with thanks to Mr Thiel
Name:
Creating a Fingerprint from DNA Evidence
In this activity you will perform electrophoresis on purified DNA samples that were collected
from a crime scene at Ardingly College. At the scene Mr. Naughton was found brutally beaten
and bleeding along the back of his head. Apparently the cowardly attacker approached Mr.
Naughton from behind as he left his room in the early evening. This hideous predator hit him
with a blunt object that left impressions in the scalp of Mr. Naughton. The pattern of the
impressions revealed what appear to be rows of key pads.
DR SPIERS – SUSPECT 1
You are interning for the Ardingly
CSI team and were called to the
scene of the crime. After several
hours of carefully collecting
evidence from the crime scene you
find a calculator in the bushes that
has blood on the key pad side of the
calculator and a small amount of
flesh in the groove of the battery
MR CROOK – SUSPECT 2
compartment on the other side,
bingo.
The DNA of the blood matches Mr. Naughton’s DNA but the DNA in the flesh sample does
not. You conclude that the flesh came from the perpetrator's hand as he committed this
heinous act.
It is a well known fact that Ardingly Science teachers, Mr. Crook and Dr Spiers, have been very
jealous of the attention that the Ardingly Biology department continually receives.
Students continually over hear them making negative comments about both Mr. Lewis and Mr.
Naughton. Concerned students have noted that both of these teachers are currently unable to
find their personal calculators. You believe that either one of these dastardly despicable
teachers could be the perp. You have requested the CSI team to take tissue samples for DNA
comparison to the flesh taken from the calculator. Both spineless teachers were forced to give
up tissue samples. Dr Spiers’ DNA was cloned in a PCR machine and put into blue reaction
vessels marked S1. Mr Crook’s DNA was cloned using PCR and placed into purple reaction
vessels labelled S2.
Your job is to perform electrophoresis on these products and compare the suspects DNA with
the product from the crime scene and determine which teacher may be the perpetrator of the
crime.
Adapted by GJL from Amador Valley High School, with thanks to Mr Thiel
Name:
1. Remove your digested
DNA samples and tap each sample on the desk to mix contents.
2. Using a separate tip for each sample try and pipette approximately
5ul of loading dye “LD” into each tube the first line on the pipette tip is
2ul and the second is 10ul. To help with the pipette technique, draw
up the pipette slightly before removing any of the dye. Ensure you
place the dye into the bottom of the sample. Cap the tubes and mix by
gently flicking your finger.
3. Place an agarose gel in the electrophoresis apparatus. Fill the electrophoresis chamber
with TAE buffer to cover the gel; using approximately 275ml the buffer must cover the
entire gel. Ensure you do not move your gel after this.
4. Using clean separate tips for each sample, load the approximately the indicated
volume of each sample into one of the four wells of the gel in the following order:
Lane1: M DNA size marker, 5ul
Lane2: CS green(Crime Scene Evidence) 10ul
Lane3: S1 blue (Suspect 1 DNA) 10ul
Lane4: S2 purple (Suspect 2 DNA) 10ul
5. At either end of the gel insert a carbon conductor sheet and then clip the black
crocodile clip to the carbon paper at the well end of the gel and clip the red crocodile
clip to the carbon paper at the opposite end of the wells.
6. Turn on the power and you will need to run the samples for the rest of the lesson
Lesson 2
1. Add 60ml of DNA stain to the tray. Cover the tray with a plastic wrap. Let the gel stain
overnight, with shaking for best results
Questions:
*11. What is a probe and what is its function when performing a Southern Blot?
12. Why were five probes used when making the DNA fingerprint?
Copy the address shown here into the location window of your browser or if already at a
computer simply click on the link.
http://www.pbs.org/wgbh/nova/sheppard/labwave.html
Do the interactive DNA fingerprinting located at the web address. This activity is not bad but it
does have some inaccuracies as a result of the limitations of the animators.
Adapted by GJL from Amador Valley High School, with thanks to Mr Thiel
Name:
13. Identify and briefly describe at least one inaccuracy
Lesson 3
1- Look at your stained gel on a light box or overhead projector. Analyze the results and
answer the questions below.
Questions:
14- Draw your gel showing your results!
15- Which "suspect" DNA matches the evidence DNA?
16- Compare your gel with others. How can you account for differences in separation and band
intensity?
17- What would happen to the separation of the bands if the agarose concentration in the gel
was increased?
18- If the two suspects were identical twins would this procedure enable you to ID the perp?
Explain your answer.
Adapted by GJL from Amador Valley High School, with thanks to Mr Thiel
Name:
Adapted by GJL from Amador Valley High School, with thanks to Mr Thiel