LAB: DNA Fingerprinting/Simulation
BACKGROUND:
The linear sequence of DNA bases contains the instructions for the
proteins that make each organism unique. A single substitution of a base - an
adenine instead of a cytosine for example - can change the entire meaning of the
code, resulting in a different protein, and sometimes, a different individual.
Restriction enzymes cut DNA. Each restriction enzyme recognizes a specific
group of "target" base pairs and makes a cut within this area.
A DNA molecule containing several such targets will be cut into a number of
fragments. The DNA from one individual will always give the same pieces
when cut with a specific enzyme because all of that person's DNA is the same.
Conversely, DNA molecules from different individuals may give different-sized
fragments. These fragments are called restriction fragment length
polymorphisms - RFLPs ("riflips") for short.
Gels are used to separate the fragments. The gel (much like jello!) offers a
matrix through which the cut pieces of DNA can move under the influence of an
electric field. The pieces of DNA, which are negatively charged, will be attracted
to the positive end of the electric field. The fragments move through the gel at
rates relative to their size, small ones migrating to the positive end the most
rapidly.
In this lab we will be using a model to demonstrate what is happening at
the molecular level in the process of DNA fingerprinting. You will be given
sentences that represent DNA samples taken from different individuals. Each
letter represents one of the four bases in the DNA sequence. Your scissors, under
your direction, will represent restriction enzymes that can cut the DNA at
defined spots. Your graph paper will represent the gel through which your
pieces of DNA will migrate. The goal is to demonstrate the technique used in
actual DNA fingerprinting.
LAB: DNA Fingerprinting/Simulation
OBJECTIVES:
� To cut three meaningful phrases (representing the DNA from three
different criminal suspects) using two different "restriction enzymes."
� To construct a model of a "gel" that demonstrates the separation of
fragments by size.
� To determine which of three suspects was at the scene of a crime by
interpreting the gel results.
PROCEDURE:
1. Separate the three phrases by cutting them out with scissors. Leave a little
trim around each. Keep the red and green phrases separate from each other.
2. Our two hypothetical restriction enzymes, X and Y, recognize the target
sequences CAR and FAST respectively.
Enzyme X cuts CAR at C / AR
Using your scissors to represent enzyme X, cut the red paper strips at all CAR
sites (all CAR sequences within a phrase should be cut). Keep each phrase in a
separate pile. You should have three piles of red phrases.
Enzyme Y cuts FAST at FAS / T
Using your scissors to represent enzyme Y, cut the green set of paper strips at all
of the FAST sites. Again, keep each phrase in a separate pile. You should have 3
piles of green phrases.
3. The graph paper represents the gel. The vertical axis is partially labeled for
you, with zero at the top and 30 at the bottom. Fill in the remainder of the
vertical axis with the appropriate numbers between 0 and 30. The vertical axis
measures the length of a fragment (the number of letters a particular fragment
contains). The distance a fragment moves in a gel corresponds to its length. The
shortest piece, which travels most rapidly, will be closest to zero at the top.
LAB: DNA Fingerprinting/Simulation
PROCEDURE (Continued):
4. On the side of the graph paper with "Enzyme X" labeled at the top, begin to
load the "gel." That is, arrange the red fragments from the meaningful phrase
"WALK FAST AND SNARE THE RAT' into the "well" marked LANE 1. "Turn
on the current and let your gel run." Move the fragments by counting the
number of letters in each fragment (count spaces between words or partial words
as one letter; do not count spaces before the first letter of after the last letter in a
fragment). Find the matching numbers on the vertical axis and paste the
fragments in place across from them. Paste the fragments so that they are more
or less centered within the lane.
5. On the same side of the graph paper, load the fragments from the red
phrase "TALK FAST AND SCARE THE CAT" in Lane 2 and the red phrase
"WALK FAST AND SHARE THE CAR" in Lane 3. Repeat moving them in the
gel as in step 4. Paste the fragments in place.
6. Below the well for Lane 1, write "Suspect 1." Below Lane 2, write "Suspect
2," and below Lane 3, write "Suspect 3."
7. Turn over the graph paper and repeat steps 4, 5, and 6 for the three sets of
green fragments.
8. I will provide you with the results of the digests done in the forensics lab
using the DNA found at the scene of a crime. Lane 1 contains crime scene DNA
cut with enzyme Y, and Lane 2 contains crime scene DNA cut with enzyme X.
LAB: DNA Fingerprinting/Simulation
ANALYSIS OF RESULTS:
Staple your graph paper (results of the simulation) to a separate piece of paper.
Answer the following questions on that separate piece of paper.
1.
Which individual was at the scene of the crime? Explain how you came to
your conclusion.
2.
What do the scissors represent in this simulation?
3.
What does the graph paper represent in this simulation?
4.
Explain the relationship between the different fragment sizes and the
distance traveled (migration distance) on the gel. Propose a mechanism for this
relationship.
5.
In the process of identifying the suspect who had his or her DNA at the
crime scene, were enzymes X and Y equally useful? Explain.
6.
In general, would using both restriction enzymes X and Y on individual
samples improve our ability to match crime scene DNA with that of a suspect?
Why or why not?