Which Genotypes Can be Matched between DNA Found at a Crime Scene and

Four Suspects Using PCR and Gel Electrophoresis?

Zahin Hussain
Mara Quinn
Hannah Schell
Zehn Wani
Alex Barbolovici

Background Information:
DNA fingerprinting is the use of STRs, or short tandem repeats of DNA, that
have undergone PCR, or polymerase chain reaction. STRs are unique to each
individual, excluding identical twins, and are used in DNA fingerprinting to
identify criminals during trials and the like. Polymerase chain reaction is used
to amplify strands of DNA in gel electrophoresis. PCR has three main steps:
Denaturation, Annealing, and Extension. Denaturation involves the heating
of DNA to break apart the hydrogen bonds that form the double stranded
DNA molecules. Annealing is when primers are binded to their respective
sites

on

the

molecule.

Extension

involves

DNA

polymerase

adding

nucleotides to the primed regions. Following PCR, the DNA molecules are put
through the process of gel electrophoresis, which is the separation of unique
DNA fragments that form unique banding patterns as they move through the
gel. This is due to the fact that DNA is negatively charged, and it is attracted
to the positive end of the gel. Larger fragments of DNA travel slower than the
short fragments because they are easily caught in the gel, allowing for the
separation by size.This allows scientists to identify who DNA belongs to, and
in effect determine the perpetrators in crimes.
Question and Purpose:
Who is the suspect that was present at the crime scene based on the PCR
amplified DNA sample and subsequent Gel Electrophoresis data?

 The purpose of this experiment is to determine which suspect was at the

crime scene by first using PCR to amplify the DNA sample and then Gel
Electrophoresis to pinpoint which suspect was at the scene of the crime.

Hypothesis and Rationale:

Hypothesis: If PCR and Gel Electrophoresis are used with DNA found at a
crime scene, then the DNA fragments will express the same distance in Gel
Electrophoresis as the corresponding suspects DNA, and the suspect will be
confirmed to be at the crime scene.
The experiments independent variables was the specific locus that was
being tested. The experiments dependent variable was the distance that the
fragments of each DNA traveled, or more specifically the length of the
fragments of DNA. The controlled variables of this experiment would be the
amount of DNA used, amount of master mix used, and the total time in gel
electrophoresis and PCR.
Materials :
Part 1: PCR

Ice bath
5 tubes of DNA (from Crime Scene and Suspect A - D)
Master Mix + primers
5 PCR tubes
5 PCR adapters
Marking pen
.5 - 10 L micropipet
1 rack of .5 - 10 L pipet tips, aerosol barrier

Part 2: Gel Electrophoresis

3% agarose gel
5 PCR samples from Part 1
350-500 mL 1X TAE running buffer
60L Orange G loading dye
25 L Allele Ladder (orange liquid)
.5 - 10 L micropipet
1 rack .5 - 10 L pipet tips, aerosol barrier
Gel electrophoresis chamber
Power supply

 Procedure (Numbered) and Diagram:

Day 1 Procedure: PCR
1. Get all necessary materials--MMP solution and tubes labeled CS, A, B, C, and
D. Obtain five tubes, label them appropriately with CS, A, B, C, and D.
2. Keep tubes on ice as much as possible during procedure.
3. Pipet 20 microliters of each solution into their respective tubes. Be sure to
use a fresh tip for each solution.
4. Pipet 20 microliters of the Mastermix + primers solution into each pipet. Be
sure to use a fresh tip for each solution. The solutions should all appear
to be blue.
5. Place capped tubes into PCR thermocycler and run the machine.

6. Diagram:
Day 2 Procedure: Gel Electrophoresis
1. Set up your MINI-One machine as instructed, be sure that plastic tray clicks
in.
2. Make the Gel:
a. Melt gel cubes in microwave for ten seconds and pour immediately into gel
tray, place teeth molds on top of gel to form grooves in gel
b. Allow gel to set
3. Place gel into MINI-One machine and pour buffer into tray, only enough to
cover the two electrodes in the tray.
4. Turn light in machine on low, place cover over machine, and start machine.

Statement of Confidence:
All pipet measurements are accurate to within .1 L.
The Buffer measurement is accurate to within .1 mL
Analysis and Summary (with picture):
We cannot iterate the specific number of base pairs in each fragment
because the Gel Electrophoresis had not ran to the edge of the agarose gel,

and we do not know the base pairs associated with the Allele Ladder.
However, we can identify the length of the fragments in relation to both the
allele ladder and the other fragments. Thus, we can see if the fragments
from one suspects DNA has either fewer, the same, or more STRs than the
crime scenes DNA. The only set of DNA fragments that seemed to perfectly
match that of the crime scene was Suspect C. None of the other suspects
had both of their fragments match those of the crime scene. Suspect A had
one fragment that was the same as the Crime Scene DNA, however, both of
the fragments must match the Crime Scene in order to be certain that they
took part in the crime. Therefore, the only suspect that was at the crime
scene was Suspect C.

Conclusion:
Claim: Through the use of PCR (polymerase chain reaction) and Gel
Electrophoresis, genotypes of STRs (short tandem repeats) , from a specific
loci, found at a crime scene will be matched up with one of the genotypes of
four suspects (A, B, C, D). In this experiment, it was found that bands of the

crime scene match with those of Suspect C, revealing him or her to be at the
crime scene.
Evidence: The bands are measured by distance travelled toward the positive
node in centimeters. As a point of reference, DNA fragments with less STRs
travel farther down the Agarose gel, than DNA fragments with more STRs.
Here are the measurements for each sample: for the crime scene, the larger
DNA fragment traveled about 0.9cm and the smaller one traveled about
1.5cm. The distances were 0.6cm and 1.5cm for Suspect A, 1.2cm and 1.8
for Suspect B, 0.9cm and 1.5cm for Suspect C, and 0.8cm and 1.9cm for
Suspect D, respectively. The DNA fragments of Suspect C were found to
travel the exact same distance as the fragments of the DNA found at the
crime scene.
Reasoning: Since the distance traveled by the bands for both the crime
scene and Suspect C were the same, there is enough evidence to suggest
that Suspect C was at the crime scene. Since STRs vary so greatly from
person to person, matching of two genotypes at a single locus through Gel
Electrophoresis provides sufficient proof that it is the DNA of the same
person. The bands for both the crime scene and Suspect C traveled 0.9cm
and 1.5cm toward the positive node using the Gel Electrophoresis,
meanwhile, the bands for the other suspects showed significant variance
from that of the crime scene in the highlighted bands, removing them from
suspicion. The bands would not be so easily visible without completing the
PCR process beforehand. Through this, the targeted DNA was replicated and
multiplied, making the bands for Gel Electrophoresis thicker.
Limitations and Sources of Error:
Changes in classroom environment - The pressure and temperature of the
classroom may have affected results and thrown off the data slightly. The
samples were not kept on ice at all times as they needed to be modified.
Contamination - The samples may have been contaminated by foreign DNA
from the air or from contaminant organisms.

 Mutation - The DNA sample used was replicated many times and this means
that mutations may have occurred as they do when many replications occur.
Mechanical problems - The mini-one system did not work the first few times
and the gel had to be moved from machine to machine and this disturbance
may have induced error.
All of these problems alone may not have been enough to alter the data but
together they may have affected the results negatively.
Sources:
Campbell, Neil A., Jane B. Reece, Lisa Andrea. Urry, Michael L. Cain, Steven
Alexander. Wasserman, Peter V. Minorsky, and Rob Jackson. "Chapter 20 :
Biotechnology." Biology : AP Edition. San Francisco: Pearson, Benjamin
Cummings, 2008. N. pag. Print.
McCune, Andrea, comp. AP Bio - PCR/Gel Electrophoresis/DNA Fingerprinting
Lab. 22 Jan. 2016. Lab rubric, background information, and procedure list for
two-day lab. Athens High School, Troy, MI.