Using VNTR analysis to Identify Guilt in at a Crime Scene

Rachel M. Lytle

High School of Telecommunication Arts and Technology

Summer Research Program for Science Teachers

Summer 2006

Subject: AP Biology/ Forensics

Grade Level: 9^th through 12^th

Unit: Genetics, Biotechnology, lab skills

Objective: Students will be able to:

• Isolate human DNA from cheek cells
• PCR amplify Human DNA. 
• Compare the process of DNA Replication and PCR
• List the steps involved in the PCR process
• Interpret gel electrophoresis gels do determine guilt in a simulated crime
• Identify the guilty suspect in a crime scenario and defend their choice with evidence. 
• Calculate the probability of obtaining a particular genotype
• Become familiar with the following terms:

denaturation, primers, replication, template, polymerase, PCR, gel electrophoresis, VNTR, autoradiogram

Prior Knowledge: Teacher discussion on DNA replication, protein synthesis and the function and structure of the DNA molecule, gel electrophoresis, pipeting skills, solution preparation. 

New York State Science Standards

Standard 1 Key Idea 1-performance indicator 1.1b

                  Key Idea 2-performance indicator 2.2a

Standard 4 Key Idea 2-performance indicators 2.1a, 2.1b, 2.1c, 2.2c, 2.2d, 2.2e 

                  Key Idea 5- performance indicators 5.1f, 5.1g

National Science Learning Standards met by this lesson:

• Content Standard A:  As a result of activities in grades 9-12, all students should develop abilities necessary to do scientific inquiry and understanding about scientific inquiry
• Content Standard B:  As a result of activities in grades 9-12, all students should develop understanding of the structure and properties of matter, chemical reactions, motions and forces, and interactions of matter and energy

• Content Standard C:  As a result of activities in grades 9-12, all students should develop understanding of the molecular basis of heredity, and matter, energy, and organization in living systems

• Content Standard E:  As a result of activities in grades 9-12, all students should develop abilities of technological design and understandings about science and technology
• Content Standard F:  As a result of activities in grades 9-12, all students should develop understanding of science and technology in local, national, and global challenges
• Content Standard G:  As a result of activities in grades 9-12, all students should develop understanding of the nature of scientific knowledge

Background Information:

A tandem repeat is a short sequence of DNA that is repeated in a head-to-tail fashion at a specific chromosomal locus. Tandem repeats are interspersed throughout the human genome. Some sequences are found at only one site -- a single locus -- in the human genome. For many tandem repeats, the number of repeated units varies between individuals. Such loci are termed VNTRs.

In diagram below one person has inherited the allele for  21 copies of a repeated sequence from its mother and 15 copies of the same sequence from its father.  

When profiles from a single VNTR locus from unrelated individuals are compared, the profiles are normally different. However it is possible for two individuals to have the same profile at one or two loci by chance. But the chance of more than one person having the same DNA profile at 4, 5, or 6 different VNTR loci is extremely small. When DNA

Lab part A

Day 1- Isolate your DNA from cheek cells,

Day 2- Set up PCR reaction

Day 3—Cast Agarose gel

Day 4- Run Gel Electrophoresis

Day 5- Photograph gel and analyze data

Day 6- Begin Probability Activity

Day 7- Complete Probability Activity

Day 8- Write recommendation to the Dean

Materials Needed

Thermocycler or 3 water baths

Centrifuge for 15mL tubes

Microcentrifuge

Hot Plates and 600 ml beakers

Adjustable micropipette

Electrophoresis chamber

Power supply

UV transilluminator

Camera to picture gel

Test tube racks

Supplies per class

Paper cup for each student

15 ml tube for each student

1.5 ml tube for each student

PCR tube for each student

Micropipets

Permanent lab markers

Reagents

300ml- 0.9% NACl solution

Chelex

MgCl2

Distilled water

Agarose

Electrophoresis buffer

Loading dye

Ethidium bromide

Teacher Preparation prior to lab

1. Add 10 mL sterile NaCl solution to 15 ml tubes
2. Add 700 ml Chelex to 1.5ml (small) tube
3. Prepare PCR Master Mix containing 500uL PCR mix, 250uL MgCl2, 375 uL distilled water- Add 45uL of this preparation to PCR tubes
4. On Day 1- prepare a boiling water bath in a 600mL beaker on a hot plate at the beginning of class. 

Student Procedure

Part 1- Isolating Your DNA from Cheek Cells

1. Use a permanent name to write you assigned lab ID # on one 15 mL tube of NaCl and two (2) 1.5 mL microcentrifuge tubes
2. Pour the saline solution into your mouth and swish for 30 seconds. 
3. Spit the saline solution into a paper cup. 
4. Carefully pour mixture from paper cup into 15mL tube
5. Take your DNA sample to the centrifuge.  The teacher will balance and load the centrifuge.  The tubes will be centrifuged at 1000xg for 10 minutes. 
6. Pour off the supernatant being careful not to disturb the cell pellet at the bottom of the tube. 
7. Obtain microcentrifuge of Chelex.  Set micropipette to 500uL.  Draw Chelex solutions into and out of pipet tip 5 times to suspend resin beads. 
8. Add 500uL of Chelex suspension to your cell pellet.
9. Resuspend cells in Chelex by pipeting in and out several times. 
10. Transfer 500uL of cells in Chelex to a clean labeled 1.5mL tube. 
11. Place your sample in a boiling water bath for 10 minutes.  Use float with handle to hold your microcentrifuge tube. 
12. Remove samples from boiling water and cool on ice for 2 minutes. 
13. Bring you sample to the microcentrifuge where the teacher will load the class samples in a balanced fashion.  Spin cell for 1 minute
14. Use a fresh pipet tip to transfer 200uL of supernatant (clear liquid not debris at bottom of tube) to clean 1.5mL tube. 
15. Store your DNA sample in the freezer until next class period.

Part 2: Setting up your PCR- making copies of your DNA for analysis

1. Use a permanent marker to label the cap of a PCR tube
2. Add the following reagaents to your PCR tube

> This solution contains 1uM of each D1s80 primer, the 4  dNTP’s (A,T,G,C) and Taq polymerase

3. Close the cap of your PCR tube and tap on the table to collect all reagents at the bottom of the tube
4. Bring you PCR tube to the teacher to load into the Thermal cycler  to run for 30 cycles at the following conditions

Questions

The sequence of the D1S80 Primers is given below.  Right the DNA sequence they will bond complimentary to. 

Primer 1

5’-GAAACTGGCCTCCAAACACTGCCCGCCG-3’

Primer 2

1. Explain why Taq polymerase is used to : "copy" human DNA in PCR and not human polymerase.

2. Describe 3 ways in which PCR is similar to DNA replication. 

3. Describe 3 ways in which PCR is different than DNA replication. 

Part 3: Cast 2% Agarose Gel- One gel will be made for every 8 students

1. Prepare a 2% agarose gel in 1X TBE to make a total volume of 100 mL.- Prepare this in a 500 mL flask
2. Once your agarose solution has been melted in the microwave (no more than 2.5 minutes on high)  ** Use gloves and be careful the solution is very hot** Let your flask cool on the bench top.  You teacher will be around to add 5uL of ethidium bromide.>

3. Wear gloves and pour your gel into the gel casting tray. 
4. Insert gel comb and allow the gel to solidify.
5. Once the gel has solidified place the gel casting tray into the gel electrophoresis chamber and add enough TBE to cover the gel.

Answer the following questions about DNA gel electrophoresis

1. In a DNA gel where will the shorter bands be found?  Where will the larger bands be found?
2. Do you want to use a higher or lower gel concentration to examine large fragments of DNA?  Explain your reasoning. 

Part 4- Run your PCR amplified DNA samples using Gel Electrophoresis

1. Obtain your PCR products from the freezer. 
2. Add 2 uL of loading dye to your sample
3. Load 20uL of 123 Base Pair Ladder in lane number 1 and lane number 6
4. Add 20uL of your sample to one lane- share the gel with other students.  Each student should use their own lane. 
5. Record the lane number you load our DNA sample into here _________________
6. Place the lid on the electrophoresis chamber and connect the apparatus to the power supply. 
7. Run the gel at 60 Volts for 30 to 40 munutes. 

Part 5-Visualize Gel

1. Using the digital camera take a picture of your gel under UV light
2. Begin the Results and Discussion portion of the lab part A, VNTR analysis of human DNA. 

Results and Discussion

1. Examine photograph of the gel containing your experiment.  Orient the photograph so the wells ae at the top.  Use the key below to help you interpret your results. 
   1. A band that appears at the bottom of the gel in the same position in each lane is no amplified DNA.  It represents a “primer dimmer” where primers amplified themselves through the PCR process. 
   2. No bands visible.  This is due to either an error during sample preparation. 
   3. One band is visible.  The person has inherited identical alleles from each parent. 
   4. Two bands visible.  The person has inherited two different bands one from each parent. 
   5. Three or more bands visible.  The primers amplified nonspecific regions of DNA.  Just look at the two brightest bands. 

Briefly describe below what information you gained from examining your gel.

2. Looking at your gel how many different alleles can you identify for the D1S80 loci?
3. What percent of the your classmates are heterozygous for D1S80 alleles?  What percent are homozygous?
4. What allele appears to have the highest frequency in the class population?
5. Do you feel that this information could be used to link a suspect to a crime scene?  Why or Why not?

At 3:01PM on October 23 of this year Mr. Michael the school’s meanest math teacher was staying after school to grade tests.  Many students did not like Mr. Michaels because he assigned tons of homework and gave his students the toughest test.  You are two other students were sitting in the room completing a test you had missed.  Mr. Michael’s wife called his cell phone so he stepped out into the hallway to use his phone.  When Mr. Michael returned he found a large glob of spit floating in his coffee.  Anxious to catch the culprit Mr. Michael took you, the two other students, and the coffee with spit in it to Mrs. Lytle’s science classroom.  Mrs. Lytle agreed to help Mr. Michaels determine who spit in his coffee using VNTR analysis.  The audioradiograms Mrs. Lytle made using all the DNA samples have been provided to you.  Using the data provided calculate the probability at which you, and the two other students could present with such a DNA profile.  Present your findings to the school dean so that he can take proper disciplinary action. 

At this point we do not know whether this is very strong evidence that the suspect committed the crime. We need to know whether such a match is likely to have occurred simply by chance, or whether it is reasonable to conclude that the multilocus genotype matches because the evidence was in fact left by the suspect. So we will calculate the probability of such a five-locus genotype occurring by chance in the population.

We will use a process called “binning”. In human DNA, because there are so many different sized alleles, some of which are very similar in size, it would be impossible to try to distinguish every different allele size on a gel of this kind. To get around this problem, bands that fall between adjacent markers are classified as all belonging to the same “bin”, even if they differ somewhat in size.

You have been given tables of actual allele frequencies from a Caucasian reference population for the three different VNTR loci used in this case, presented on the same page with the autoradiogram showing the pattern observed at that locus. These tables show the alleles sizes that were observed when these loci were scored in large samples of individuals. You can see that the number of possible combinations of alleles is very large.

The column labeled “between markers” tells you the size class (“bin”) of the alleles, the column labeled “count” tells you the number of people in the sample population who had that allele. And, the column labeled “fraction” tells you the frequency of that bin in the sample.

INSTRUCTIONS: After you run your gel looking for your alleles at the D1S80 locus shade in the appropriate box to facilitate your probability calculation. 

D1S80 Locus

Before you begin your probability calculations take a careful look at all 5 autoradiographs provided to you. 

Describe any trends you notice below. 

Now you need to examine each autoradiograph and determine the frequency of the genotype for each allele for the 3 students present at the crime scene.  Place your work in Table 1, Table 2, and Table 3. 

Once you are determined the probability of each genotype you are ready to determine the probability of each person having the specific genetic make-up when multiple alleles are considered.  Record your work in table a, table b, and table c. 
 

Examine the autoradiogram on the left.  Identify the alleles present in each sample and the frequency at which each allele should appear in a population by using the data provided on the right.  Record you information in the Data Table 1

Examine the autoradiogram on the left.  Identify the alleles present in each sample and the frequency at which each allele should appear in a population by using the data provided on the right.  Record you information in the Data Table 1

Examine the autoradiogram on the left.  Identify the alleles present in each sample and the frequency at which each allele should appear in a population by using the data provided on the right.  Record you information in the Data Table 1

Examine the autoradiogram on the left.  Identify the alleles present in each sample and the frequency at which each allele should appear in a population by using the data provided on the right.  Record you information in the Data Table 1

 TABLE 1

According the rule of multiplication, the probability of two or more independent events occurring in combination is the product of the two or more independent event probabilities.  Calculate the probability for the multi-locus genotype for two loci.  Do this by multiplying together the frequencies of each of the two loci. 

TABLE A

TABLE 2

According the rule of multiplication, the probability of two or more independent events occurring in combination is the product of the two or more independent event probabilities.  Calculate the probability for the multi-locus genotype for two loci.  Do this by multiplying together the frequencies of each of the two loci. 

TABLE B

TABLE 3

According the rule of multiplication, the probability of two or more independent events occurring in combination is the product of the two or more independent event probabilities.  Calculate the probability for the multi-locus genotype for two loci.  Do this by multiplying together the frequencies of each of the two loci. 

TABLE C

Results and Discussion

1. Who spit in Mr. Michael’s coffee?  With what certainty do you know?

2. The school’s science budget has been cut.  We need to perform this genetic test using only one VNTR loci.  Which loci would be the best on to use if you could only use one loci?

3. Write a formal letter to the school Dean informing him of your findings.  Recommend which student should be disciplined and how they should be disciplined. 

4. At least 9 former death row inmates in the US have been exonerated because of DNA testing.  How many loci do you think should be required to convict a person of a crime in the court of law?  Provide evidence for your reasoning. 

References

1. http://www.zoo.utoronto.ca/able/volumes/copyright.htm
2. http://www.zoo.utoronto.ca/able/volumes/vol-15/1-bloom.pdf#search=%22dna%20and%20d1s80%20and%20bloom%22
3. http://cibt.bio.cornell.edu/labs/mb.las
4. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=50614