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DNA Fingerprinting
Can DNA evidence solve crimes?
Introduction
DNA fingerprinting is now used routinely to solve crimes. In recent years, news stories
have reported how miniscule amounts of DNA have been used to identify individuals
involved in incidents even many years in the past. It has also been used to exonerate
innocent people from incrimination. The power of DNA as a tool for individual
identification is known as DNA fingerprinting.
Like the fingerprints that came into use by detectives and police labs during the 1930s,
each person has a unique DNA fingerprint. Unlike a conventional fingerprint that occurs
only on the fingertips and can be altered by surgery, a DNA fingerprint is the same for
every cell, tissue, and organ of a person. It cannot be altered by any known treatment.
Consequently, DNA fingerprinting is rapidly becoming the primary method for
identifying and distinguishing among individual human beings.
An additional application of DNA fingerprint technology is the diagnosis of inherited
disorders in adults, children, and unborn babies. The technology is so powerful that, for
example, even the blood-stained clothing of Abraham Lincoln could be analyzed for
evidence of a genetic disorder called Marfan's Syndrome.
DNA typing is used in forensics, anthropology and conservation biology not only to
determine the identity of individuals but also to determine relatedness. This process has
been used to free innocent suspects, reunite children with their relatives, identify stolen
animals and even used in times of war to identify the remains of soldiers killed in
combat.
The Structure of DNA
The characteristics of all living organisms, including humans, are essentially determined
by information contained within DNA that they inherit from their parents. The molecular
structure of DNA can be imagined as a zipper with each tooth represented by one of four
letters (A, C, G, or T), and with opposite teeth forming one of two pairs, either A-T or G-
C. The letters A, C, G, and T stand for adenine, cytosine, guanine, and thymine, the basic
building blocks of DNA.
The information contained in DNA is determined primarily by the sequence of letters
along the zipper. For example, the sequence ACGCT represents different information
than the sequence AGTCC. The traits of a human being are the result of information
contained in the DNA code.
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Living organisms that look different or have different characteristics also have different
DNA sequences. The more varied the organisms, the more varied the DNA sequences.
DNA fingerprinting is a very quick way to compare the DNA sequences of any two
living organisms.
Even more than a finger's print, each person's "DNA fingerprint" is unique. While old-
fashioned fingerprints record the wavy, whorled ridges on fingertips, DNA fingerprints
record the chemistry of small sections of DNA in chromosomes. A genome is a complete
set of human chromosomes. It is a string of DNA with about 3 billion bases, divided
into roughly 35,000 genes. Still, the DNA in every one of your cells codes for all of the
thousands of proteins you need from conception to adulthood (DNA's entire job is to
serve as a pattern for proteins.)
For instance, an intron might have the sequence "CGGT" and it might form the repeat
"CGGT CGGT CGGT CGGT CGGT CGGT." This number varies in different
individuals and this variability accounts for the unique fingerprint in each and every
organism. There are many introns present in DNA and these regions can be analyzed to
determine a fingerprint for every individual.
Objectives:
You should be able to:
1. Run a gel electrophoresis and to separate DNA fragments
2. State or explain the basic principles involved in DNA fingerprinting
3. Analyze the data generated in this investigation
4. Describe the methodology of the investigation
5. Use evidence to solve a crime
Making DNA Fingerprints
DNA fingerprinting is a laboratory procedure that requires several steps:
1: Isolation of DNA. DNA must be recovered from the cells or tissues of the body. Only a small
amount of tissue - like blood, hair, or skin - is needed. For example, the amount of
DNA found at the root cells of one hair is usually sufficient. The procedure is
similar to the one you used when extracting DNA from the onion.
DNA Profiling can be performed on:
Chewing gum
Licked stamps
Hair samples (with roots intact)
Razor shavings
Toothbrush
Cigarette butts
Body fluids like blood and semen
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2: Cutting this DNA. Special enzymes called restriction enzymes are used to cut the DNA at specific
places. For example, an enzyme called EcoR1, found in bacteria, will cut DNA
only when the sequence GAATTC occurs. If the restriction site occurs in more
than one location on a DNA molecule, a restriction enzyme will make a cut at
each of those sites, resulting in multiple fragments.
If a given linear piece of DNA is cut with a restriction enzyme whose specific
recognition code is found at two different locations, how many fragments will be
produced? ___________________
Restriction Enzymes: Molecular Scissors
A restriction enzyme is used then to cut out the targeted gene from that chromosome.
Restriction enzymes are proteins that cut DNA at specific sites. There are thousands of
restriction enzymes and each is named after the bacteria from which it was isolated.
Three common restriction enzymes are EcoR1, Hind III, and PstI. EcoRI was isolated
from Escherichia coli bacteria, Hind III was isolated from Haemophilus influenzae
bacteria, and PstI was isolated from Providencia stuartii bacteria. Each restriction
enzyme recognizes a specific nucleotide base sequence in the DNA, called a restriction
site, and cuts the DNA molecule at only that specific sequence.
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In general, restriction sites are palindromic, meaning they read the same sequence of
bases forwards and backwards on the opposite DNA strand.
For example, in the figure below, GAATTC read to the right has a counterpart in the
lower strand, CTTAAG read to the left. These are called palindromic sequences.
An important feature of restriction enzymes is that each enzyme only recognizes a
specific palindrome and cuts the DNA only at that specific sequence of bases. A
palindrome can be repeated a number of times on a strand of DNA, and the specific
restriction enzymes will cut all those palindromes at their restriction sites , no matter
what species the DNA comes from.
The restriction enzyme EcoRI cuts DNA between the G and the A in a GAATTC
palindrome.
How many base pairs are there to the left of the cut?
How many base pairs are there to the right of the cut?
Counting the number of base pairs, is the right fragment the same size as the left
fragment?
You can describe the fragment size by referring to the number of base pairs in the
fragment. This method is typically used to determine the size of fragments and it is
represented in base pairs.
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If the GAATTC palindrome is repeated four times on the same piece of linear DNA how
many DNA fragments would be produced upon cutting the DNA with EcoRI? _________
What have we learned so far?
1. The base sequence in one strand of DNA can have a palindrome in the other
strand.
2. Palindromes can be detected by restriction enzymes
3. Restriction enzymes cut the palindromes at restriction sites.
4. A restriction enzyme only recognizes one specific kind of palindrome
5. Cutting DNA at restriction sites will produce DNA fragments
6. Fragment size can be described by the number of base pairs they contain
3: Separating the cut DNA by gel electrophoresis
Electrophoresis separates DNA fragments according to their size. DNA
fragments are loaded into the wells of an agarose gel. Buffer is added to the
electrophoresis chamber and an electric current is applied. The matrix of the
agarose gel acts as a sieve through which smaller DNA fragments move faster
than the larger ones.
Each person has differences in DNA sequences that are unique. The DNA
isolated from individuals is cut with restriction enzymes and then run on an
agarose gel. The gel is then stained and compared to DNA obtained from other
individuals. Based upon these results, one can determine which DNA fingerprint
is similar to the one obtained at the crime scene for instance.
Agarose Gel Electrophoresis: A Molecular Strainer
How can DNA fragments be separated from one another?
Agarose gel electrophoresis is a procedure used to separate DNA fragments based on
their sizes. Electrophoresis means to carry with electricity. DNA is a negatively charged
molecule. Scientists have used this fact to design a method that can be used to separate
pieces of DNA. A solution containing a mixture of DNA fragments is placed into a small
well formed in a gel that has a texture similar to gelatin. The gel is then placed in an
electrophoresis chamber that is filled with a conductive buffer solution. An electric
current is applied to the gel. This current causes the negatively-charged DNA molecules
to move towards the positive electrode.
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You can think of an agarose gel as a strainer with tiny pores that allow small particles to
move through it very quickly. The larger the size of the particles, the slower they are
strained through the gel. After a period of exposure to the electrical current, the DNA
fragments will sort themselves out by size. Fragments that are the same size will tend to
move together through the gel and form bands. Therefore, the rate at which a DNA
fragment migrates through the gel is inversely proportional to its size in base pairs.
Over time, the smaller DNA fragments will travel farther than the larger ones. The bands
of DNA will be seen in the gel after the DNA is stained.
DNA is colorless so DNA fragments in the gel cannot be seen during electrophoresis. A
blue loading buffer, containing a blue dye, is added to the DNA solution. The loading
dyes do not stain the DNA but make it easier to load DNA into the wells of the gel. the
blue dye also allows one to see how far the DNA is moving along the gel.
Today’s Lab Exercise:
In this laboratory exercise, you will analyze three different samples of DNA. One
sample has been collected from a crime scene and the other two samples are
obtained from suspects 1 and two. The DNA from all three samples has been cut
with EcoR1 and Pst1 (see below) which are the most common enzymes used in
forensic science. The resulting DNA fragments will be separated and visualized
on agarose gels. Based on the restriction fragment patterns, you will compare the
evidence and match one of the suspect’s DNA to the sample collected at the crime
scene.
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Procedure for sample preparation and gel electrophoresis
Follow figure on following page.
1. Obtain an agarose gel and place it in the electrophoresis apparatus. Be sure to
make sure you are wearing gloves and goggles while handling the gels and
running this procedure. Do not handle gels without gloves and goggles.
2. Fill the electrophoresis chamber with 1X TBE buffer to cover the gel,
approximately 250 ml.
3. Make sure the wells are near the black electrode (-)
4. Using a separate tip for each sample, load 10 l of DNA marker in well 2 and
then load 10 l of DNA samples into the next few wells.
Lane 2: DNA marker standard
Lane 3: crime scene DNA sample
Lane 4: suspect 1 DNA sample
Lane 5: suspect 2 DNA sample
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5. Place the lid on the electrophoresis chamber. The red and black jacks on the
lid should match up.
6. Place leads into the power supply and match the red with red and black with
black.
7. Turn on the main power switch. Set the voltage to 110 volts and press the
“marathon person” button to begin the electrophoresis. Allow gel to run for 50
min.
8. Turn off the power supply and remove the top of the gel box.
9. Carefully remove the gel and tray from the gel box.
10. Analyze the results by placing your group’s gel in the light box and camera set
up as per the instructions listed below.
Gel Electrophoresis Analysis Procedure for Instructors:
1. NOTE TO INSTRUCTOR: Before beginning, prepare a PowerPoint presentation
with a slide for each of your student groups. Put the instructor name, section number and date on the first slide in the “click to add title” box. Put the names of each student in the group in the “Click to add title” section of each slide. You will be pasting the picture of the group’s gels in the “Click to add text” section of each slide.
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2. Turn on the Light Box. Lift the orange lid and place a gel on blue surface of the Light Box. Note: It is easier to position the gel in front of the camera when the light box is on.
3. Open LoggerPro software.
4. Click on the Insert option on the menu to open a drop down menu.
5. Select Gel Analysis.
6. Click on Take Photo.
7. A new window will open that will show you the live picture.
8. Position the gel under the camera so that it is centered.
9. Click the Take Photo button.
10. The window will close and the photo should appear in a new window.
11. Right click on the picture of the gel to open a menu.
12. Select Copy from the menu.
13. Open the PowerPoint presentation. Left click on the “Click to add text” portion of the slide. Click “Paste” from the left-hand side of the Home tab to past the slide into the file.
14. Repeat this process until you have pasted all of the student groups gels into the PowerPoint presentation.
15. Save the PowerPoint presentation.
16. Have copies made of the PowerPoint presentation and bring them to the next lab. You will need four copies (one for each student in each group).
17. You can also distribute the files to your students through WebStudy or via email.
18. During next week’s lab, students will use the photographed copy of their gel to complete their analysis as part of a formal laboratory report.
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Results
Here is the replica of the gel you are running:
Marker CS S1 S2
Draw a replica of the prepared gel at your bench of the DNA samples above:
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How many different sizes of the DNA fragments are in each sample?
Crime Scene _______
Suspect 1 __________
Suspect 2 __________
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Is there a suspect that matches the crime scene? ____________________
How sure are you that this is a match? ____________________________
Questions:
Number 1
The schematic represents a small section of DNA from three different individuals.
1. Compare the sugar phosphate backbone in the side chains of all three individuals. Are
there any differences?
2. Do all three samples contain the same bases? Explain.
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3. Are the bases paired in an identical manner in all three samples? What is the base
pair bonding rules?
4. What differences do you observe in the three individuals?
5. What will you need to compare between these DNA samples to determine if they
are identical or not?
Question 2:
Observe the two samples of DNA shown below:
Sample 1: CAGTGATCTCGAATTCGCTAGTAACGTT
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Sample 2: TCATGAATTCCTGGAATCAGCAAATGCA
If both samples are treated with a restriction enzyme that recognizes the sequence
GAATTC then indicate the number of fragments and the size of each fragment from each
sample of DNA.
Sample 1: Number of fragments _________________
Sample 2: Number of fragments _________________
Question 3
A piece of DNA is cut into four fragments as shown in the diagram. The solution
containing the four fragments is placed in a well in an agarose gel. Using the information
given, draw how you think the fragments might be separated. Label each fragment with
its corresponding letter.
A B C D
Complete this rule for the movement of DNA fragments through an agarose gel
THE LARGER THE DNA FRAGMENT, THE_______________________________
Question 4
Observe the diagram of the agarose gel below. It shows two lanes. Remember a lane is
the column of bands below a well. The right lane contains a banding pattern from four
fragments of known length
6000 base pairs
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5000 base pairs
3000 base pairs
1000 base pairs
1. Which lane is the reference ladder or marker? ______________________
2. How did you come up with this conclusion?
3. Label each band in the right lane with its base-pair size.
4. Compare the two columns of bands. Estimate the size of the fragments in the left
lane.
a. upper band ____________________
b. lower band ___________________
5. How did you determine the sizes of the two bands in the left lane?
Question 5:
Analyze the bands on the following gel. Answer the following questions.
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1. What is contained within each band seen?
2. If this were a fingerprinting gel, how many samples of DNA can you assume were
placed in each separate well?
3. What caused the DNA to become fragmented?
4. Which of the DNA samples have the same number of restriction sites for this
restriction enzyme? Write lane numbers.
5. Which sample has the smallest DNA fragment?
6. How many restriction sites were there in lane three?
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7. Which DNA samples appear to have been cut into the same number and size of
fragments?
8. Based on your analysis of the gel, what is your conclusion about the DNA
samples in the gel. Do any of the samples seem to be from the same source? If
so, which ones? Describe the evidence that supports your conclusion.
Question 6:
By observing this gel, who committed the crime?
Question 7:
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Based on the evidence below, what do you conclude about who committed the crime.
Explain how you came up with this conclusion
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