7. DNA Fingerprinting - Logistics

Time
Day 1+: Investigating the crime scene (may take up to a week depending on the complexity of the evidence)
Day 2: Creating DNA samples and replicating DNA
Day 3: Running the “gel” and analyzing DNA fingerprint results

Grouping
Crime scene may be studied in teams or as a whole class. DNA samples are created and replicated individually. The gel is run and analyzed as a whole class.

Materials

• Scissors
• Adding machine tape (1 meter per person)
• Meter sticks (or rulers)
• Optional: Masking tape or blue painters tape to mark off a gel, loading wells, and lanes on the floor of the classroom, gym or hallway.
• Optional: Additional materials to stage a crime scene. Be creative! For example, the kids walk into class to find the classroom pet kidnapped with a ransom note, a few human hairs (for a DNA sample) caught on the cage lid, and some fingerprints left behind. At my school, the gym teacher happily volunteered to be the culprit and all the teachers contributed an adding tape DNA sample for our analysis.

Setting
Classroom and possibly a hallway or gym to “run” your “gel”.

7. DNA Fingerprinting - Background

Teacher Background
The crux of this activity is the creation of a DNA sequence on a strip of adding tape, replication of the DNA, then using these DNA sequences to perform DNA fingerprinting.

DNA replicationThe base pairing rules help explain the process of DNA replication – how a cell makes an exact copy each strand of DNA just before it divides. First, an enzyme called helicase unzips the DNA down the middle of the ladder, in between the base pairs. Next, an enzyme called DNA polymerase reads one half of the strand, identifies a matching nucleotide, and builds a new partner strand. The process is complicated by the fact that DNA polymerase can only work in one direction along the sugar-phosphate backbone (remember, that the 2 backbones are oriented in opposite directions to one another). Thus, while DNA polymerase can easily run continuously along one strand, known as the “leading” strand, the other “lagging strand” must be assembled in a piecemeal fashion, one section at a time.

DNA fingerprinting is a technique used to distinguish between individuals of the same species using only samples of their DNA. Its invention by Sir Alec Jeffreys at the University of Leicester was announced in 1985.

DNA fingerprinting is used in forensic science, to match suspects to samples of blood, hair, saliva or semen. It has also led to several exonerations of formerly convicted suspects. It is also used in such applications as studying populations of wild animals, paternity testing, identifying dead bodies, and establishing the province or composition of foods. It has also been used to generate hypotheses on the pattern of the human diaspora in prehistoric times.

Two humans will have the vast majority of their DNA sequence in common. The traditional method of DNA fingerprinting uses restriction enzymes that cuts DNA at a specific sequence. Restriction enzymes work by recognizing a specific DNA sequence and cutting the DNA within this sequence. For instance, the restriction enzyme known as Sma1 looks for the sequence CCCGGG and cuts the DNA between the middle C and G. Other restriction enzymes make a staggered cut with some overhang on each end. For instance, the restriction enzyme EcoR1 looks for the sequence GAATTC and makes a staggered cut leaving what is known as a sticky end.

If designed correctly, a restriction enzyme can target a part of the genome that his highly variable from person to person such that some individuals’ DNA will be cut while others won’t, resulting in variable length DNA pieces. These differences are known as restriction fragment length polymorphisms. This technique is also often used to determine an individual’s genotype for a given gene – for instance, to test for the presence or absence of a mutation that confers a certain genetic disorder.

These DNA pieces may then be separated using gel electrophoresis. This method places the DNA sample in a well on one end of a sheet of agarose gel, similar to a thin layer of jello. An electric field is then applied to the gel. Since DNA is negatively charged, it is attracted to the positive end of the field and begins moving through the gel. The larger, longer fragments cannot travel as far through the gel matrix and get trapped near where the sample is loaded. The smaller, shorter fragments can wiggle their way through the matrix more easily and thus travel further. This results in a unique pattern of bands for each individual, depending on the DNA sequence.

Another common method of DNA fingerprinting exploits highly variable repeating sequences called short tandem repeats (STRs). Different people have different numbers of repeat units. For instance, the CSF gene contains the sequence AGAT repeated anywhere between 6 to 16 times. Two unrelated humans will be likely to have different numbers of this AGAT sequence.

While the variable number of repeats displayed at any single STR region will be shared by around 5 – 20% of individuals, if you look at several STR regions simultaneously, this method becomes incredibly powerful. The more STR regions that are tested in an individual the more discriminating the test becomes. In the U.S.A., there are 13 loci (DNA locations) that are currently used for discrimination. This has resulted in the ability to generate match probabilities of 1 in a quintillion (1 with 18 zeros after it) or more. Therefore, it is possible to establish a match that is extremely unlikely to have arisen by coincidence, except in the case of identical twins, who will have identical genetic profiles.

The process begins by extracting DNA from the cells in a sample of blood, saliva, semen, or other appropriate fluid or tissue. Next, each STR region is amplified using polymerase chain reaction (PCR) so that the initially tiny DNA sample is copied many times, creating a sufficiently large pool of DNA for analysis. Finally, the DNA fragments are then separated and detected using gel electrophoresis. Again, the shorter the sequence of repeats, the further the DNA fragment will travel through the gel.

Student Prerequisites
A solid understanding of DNA structure and base pairing rules.

7. DNA Fingerprinting - Getting Ready

Getting Ready

1. Day 1 - Set up the crime scene. See Sources section for some great ideas for crimes that can be staged in a classroom and ideas for how to run a forensics unit.
2. Day 2 - Cut adding tape into 1 meter long strips. Set out scissors and meter sticks.
3. Day 3 – Tape out areas for the gels. All students can run their DNA on the same gel, or you can spread them out a little more by having groups of 5 students run their DNA on separate gels. For 5 students to run a gel, tape out a 100 inch (8.33 foot) x 60 inch (5 foot) rectangle. For all students to run their DNA on the same gel, you will need an area that is 100 inches long and wide enough for each student to have a minimum of 1 foot per person. The calculations are easiest if a single base pair DNA piece can travel 99 inches, 2 base pairs travel 98 inches, and so on. If your floor has small floor tiles, you can use those as your markers rather than inches.

7. DNA Fingerprinting - Lesson Plan

Lesson Plan
Day 1+ - Investigating the crime scene
If you set up a crime scene, make lots of observations of the crime scene. Start with all the kids outside the crime scene area, drawing pictures and writing down the things they notice. Finally, allow one student at a time enter the crime scene area wearing gloves to collect evidence. Evidence should be kept in plastic bags. Analyze any non-DNA evidence first. Dust for fingerprints. Collect hair and fiber samples. Perform paper chromatography on the ransom note and compare it against the pens in the possession of the various suspects. (See Sources section below for resources and lesson plans describing how to conduct these tests.)

Day 2 – Creating the DNA sequences and replicating DNA

1. Each person should lay a meter stick down the middle of their strip of adding tape with the centimeter markings facing upward.
2. Create a strand of DNA on the adding tape by writing a letter for a nucleic acid (A, T, C, or G) every centimeter along the top of the adding tape and writing the matching base below the meter stick. Remind students that they are creating their own individual DNA sequence and every person’s DNA is different, therefore they should independently make up their DNA sequence and not copy another student’s sequence.
3. The first step of DNA replication uses scissors to represent the enzyme helicase. Helicase unzips the DNA down the middle of the DNA ladder, thus each student should cut their adding tape DNA down the middle between the bases, making sure there is white space on the paper both above and below the cut.
4. Next, DNA polymerase finds the matching base and creates a new strand alongside the old strands. Your pencil is DNA polymerase and should follow along the half strands, filling in the matching bases. If you want, use a different colored pencil to create the new strand, that way you can tell the difference between the newly assembled strand and the original strand. Similarly, if you want, instruct students to fill in the matching bases continuously from left to right on the top “leading” strand but to fill in the matching bases on the bottom “lagging” strand 20 bases at a time from right to left. (Start at base #20, then match #19, #18, and so on until you hit #1. Then start at base #40, then match #39, #38, and so on until you hit #21.)
5. Each person should end up with two exact copies of their DNA.

Day 3 – Running the “gel” and analyzing DNA fingerprint results

1. Give students a quick overview of how DNA fingerprinting works:
• collect the DNA sample
• cut the sample with restriction enzymes
• sort the pieces by length using gel electrophoresis
2. Collect one copy of each students’ adding tape DNA. These may be pinned to a bulletin board and will form the class “DNA library”. Students should keep the other copy at their desks. Students can be told that you are “extracting a DNA sample” from each student to compare against the DNA of the culprit and other suspects. The “extraction process” used in this case results in paper strips of DNA rather than real DNA like the strawberry DNA extraction.Cutting DNA at sequence -AT-
3. Briefly describe what restriction enzymes are and how they work. The DNA sample the students kept will be cut using a restriction enzyme known at “AT”. Instruct students to read through the top row of their DNA. Any time they see the sequence AT (the complementary sequence below will read TA), they should cut the DNA in between the A and the T. On average, each student will make between 6 and 7 cuts although, depending on the sequence they created, some may make no cuts and some may make 20 or more.
4. Next, students should measure the length of each DNA segment in centimeters and write this information on the back of each segment. Since each base pair was written 1 centimeter apart, the length of the DNA segment equals the number of base pairs on that segment.
5. Line students up on the starting line of the gels. Explain how smaller segments will move farther. The rule is that each piece moves 100 inches minus the length of that piece. Thus a DNA segment only 1 base pair long can travel 99 inches, 2 base pairs travel 98 inches, and so on. An uncut DNA sample will not move at all and should stay on the staring line.
6. Give students a few minutes to separate and sort their DNA pieces. When a given student is done, make sure they stand outside of the gel. It is easy to disturb DNA segments simply by walking past them.
7. When all students have finished, stand back and look at the patterns made by the DNA segments. Some questions to ask include:
• How many “bands” does each person have?
• How far did the different segments move?
• Did anyone have exactly the same pattern of bands as someone else? Why or why not?
• Could this method be used to match a suspects DNA to a DNA sample taken from a crime scene? How?
• How reliable is this method? Is it possible to have matching patterns but a different DNA sequence? Is it likely?
• Should someone be convicted solely on DNA evidence? Should someone be released if DNA evidence shows they do not match the sample taken from the crime scene?
8. Another direction to take this discussion is the ethics of maintaining a DNA library. DNA libraries generally only store information on STRs or restriction fragment length polymorphisms, not a person’s full genetic code. It is exceedingly useful in identifying offenders in situations where a DNA sample is collected from the scene of a crime. On the other hand, the existence of such a library may violate privacy rights. You could ask:
• Now that you know how DNA fingerprinting works, how do you feel about the way I collected and saved a copy of your DNA in a DNA library?
• Could a DNA library be useful in solving crimes?
• How could a DNA library be abused or misused by the police or government?
• Should governments maintain a DNA library? Why or why not?

7. DNA Fingerprinting - Assessment

Assessment

1. Watch an episode of CSI that includes DNA fingerprinting data collection and evidence. Have students compare the techniques as seen on the show to the modeled version used in the classroom.

Going Further

1. Have student run a gel in a virtual lab. The Genetic Science Learning Center at the University of Utah has a great online activity that allows you to load and run a gel
2. If you have the equipment, teach your students to load and run a gel using any extracted DNA sample (see DNA Extraction lesson). Even if you don’t have gel boxes and fancy equipment, you can create your own functional gel boxes out of grocery store materials. The best write up for DNA extraction and running a gel “MacGyver style” can be found at BioTeach.

7. DNA Fingerprinting - Sources

Sources
If you are interested in creating a full-fledged CSI experience, an indispensable resource for teachers is the book, Mystery Festival, published by the Lawrence Hall of Science.

Other forensics science resources include:

• Brian Bollone of Northpoint High School has made many of his teaching resources for his criminalistics and forensic science class available on the web.
• Court TV has a wonderful series of mysteries to use in the classroom using a huge array of different techniques: DNA analysis, gunshot residue, pH testing, shoeprints, flame tests and more to solve the crimes.
• DiscoverySchool.com has a large collection of forensic science resources for teachers.
• Susan Seagraves created a fabulous “Whodunnit?” website with lesson plans for finger print analysis, chromatography, and mor.
• The Shoder Education Foundation provides a comprehensive forensics resource for teachers including lesson plans, several mysteries, and resources.

Finally, for a great real world mystery to solve using DNA analysis among other techniques, go to the “Recovering the Romanovs” from DNA interactive by Cold Spring Harbor. It is quite simply, extraordinary.

Standards
Grade 7
Genetics
2. A typical cell of any organism contains genetic instructions that specify its traits. Those traits may be modified by environmental influences. As a basis for understanding this concept:
e.    Students know DNA (deoxyribonucleic acid) is the genetic material of living organisms and is located in the chromosomes of each cell.

Grades 9-12
Genetics
4. Genes are a set of instructions encoded in the DNA sequence of each organism that specify the sequence of amino acids in proteins characteristic of that organism. As a basis for understanding this concept:
c.     Students know how mutations in the DNA sequence of a gene may or may not affect the expression of the gene or the sequence of amino acids in an encoded protein.

5. The genetic composition of cells can be altered by incorporation of exogenous DNA into the cells. As a basis for understanding this concept:
a.    Students know the general structures and functions of DNA, RNA, and protein.
b.     Students know how to apply base-pairing rules to explain precise copying of DNA during semiconservative replication and transcription of information from DNA into mRNA.
d.    * Students know how basic DNA technology (restriction digestion by endonucleases, gel electrophoresis, ligation, and transformation) is used to construct recombinant DNA molecules.