Macro-DNA: Doing DNA Fingerprinting
and Gel Electrophoresis on a Large Scale

Michael Foley
1994 Woodrow Wilson Biology Institute

Teacher Notes

Introduction: This is a paper lab that may be used either to introduce a "wet lab" gel electrophoresis, or as a substitute for the wet lab altogether. As an introduction, it can be done to familiarize the students with the terminology required to actually do a gel electrophoresis using restriction enzymes. This allows the teacher to explain to the students what is happening on the molecular level, so that they may better appreciate the need for proper technique in performing the lab. This lab also allows teachers who lack the resources to "do" gel electrophoresis with their classes the chance to show the importance of this technique, and to allow the students to learn some of the terminology of genetic technology through a fun and thought provoking hands-on activity.

Class Preparation:

Copy the six pages of boy's and girl's DNA sequences (if possible, Xerox the boy's using blue paper and the girl's using pink paper). Cut the individual lengths apart, and place the 9 boys' sequences separate from the 9 girls' (mix and match the x's, y's and z's to get additional sequences).
Copy and cut out each of the ECO R1 Restriction Enzyme Models. Be sure each student receives one enzyme to be used in determining where the "cuts" will occur in their DNA sequence (if you laminate them, you can use them again!)
Copy a class set of the Gel Electrophoresis Sheet.

During Class:

Give each boy and each girl three strips of a given numbered DNA sequence (one X, Y, and Z), which the students will tape together in that order. Be sure to tape the sequences together in proper number order.
The ECO R1 Restriction Enzyme cuts, in reality, between the G-A on the upper row and the A-G on the lower row. To make life easier on the students, simply have them hole punch the G and A on the upper sequence. In this way, as they move the enzyme down the DNA molecule, wherever a G-A appears in the two holes, they blacken in the holes. They later will cut the DNA at the point between the blackened bases.
If you wish to be more accurate in how the restriction enzyme cuts, then also hole punch the A-G on the lower part of the ECO R1 Restriction Enzyme Model. Cutting occurs between the A-G sequence on the lower strand of DNA. This will leave the typical 3' and 5' "sticky" ends. At this point you could ask what these sticky ends could be used for (recombinant DNA!!!).
When the students count the number of bases in their RFLP's (restriction fragment length polymorphisms - DNA fragments of varying lengths produced when a restriction enzyme is able to cut at different points along the same chromosome), have them count the UPPER row of bases, not the length from one end to the other, and not just the number of paired bases. They should then shade the boxes in on their Gel Electrophoresis Sheet which represent the individual fragments.
Students can now compare their sequence to the "baby" DNA fingerprint. If you tape them across the front board and around the room, students can then see what differences there are in each other's DNA.

Variations on a Theme:

Enzyme Research: have the class use different restriction enzymes. Many DNA technology books will give a listing of the enzyme cutting sites. Note: for this set of sequences, the ECO R1 works very well; many other actual restriction enzymes may only cut at a couple of points. You might have the students develop their own "personalized" restriction enzymes and use it on two different sequences (for this reason you might have a few extra copies of the DNA sequences for students to try their custom enzymes on). Discuss the patenting of microorganisms and their organic products by the biotechnology industry.
Forensics Lab: tell the class they must use DNA fingerprinting to determine who committed the crime from a DNA sequence found in hair, skin or other tissue found at the scene. Discuss the history of DNA fingerprinting and the controversy involved in its use.
Genetic Testing for Sickle-Cell Anemia: amniocentesis is carried out on a woman whose family history has seen either the disease or the trait. Normal hemoglobin DNA has the base sequence GGTCTCCTT, and sickle-cell hemoglobin DNA has the sequence GGTCACCTT. The restriction enzyme Mst II cuts between the first T and C in the DNA sequence GGTCTCC. Give the students the following DNA sequences and have them do a paper Gel Electrophoresis:
Individual #1
A A G G T C T C C T C T A A T T G G T C T C C T T A G G T C T C C T T
T T C C A G A G G A G A T T A A C C A G A G G A A T C C A G A G G A A

Individual #2
A A G G T C T C C T C T A A T T G G T C A C C T T A G G T C T C C T T
T T C C A G A G G A G A T T A A C C A G T G G A A T C C A G A G G A A
The Mst II enzyme cuts individual #1's sequence (normal hemoglobin) into four RFLP's of 5, 6, 10, and 14 units of length. Mst II cuts individual #2's sequence (sickle-cell hemoglobin) into three RFLP's of 5, 6, and 24 units. An alternate assignment might be to give a Gel Electrophoresis Sheet with 25 lines, and several individuals DNA sequences, and give the DNA fingerprints for a normal fetus, a sickle-cell anemia fetus, and a mix of the two (without giving them who is who), only the above highlighted information. Ask the students to role play as a genetic counselor and require them to decide what they will say to the parents in all three cases. (Credit for the sickle-cell lab idea comes from Judith Averback, at the July 1994 Woodrow Wilson High School Biology Institute).

References:

Drexler, David, John Hinchee, et. al. 1989. Advances in Genetic Technology. DC Heath and Company

Jenkins, Christie L. April 1987. "Recombinant Paper Plasmids." The Science Teacher.

Micklos, David A . and Greg A. Freyer. 1990. DNA Science. Cold Spring Harbor Press.

Waterman, Howard, and C. Hammond. 1990. DNA Restriction Fragment Length Polymorphisms (A Classroom Simulation). National Association of Biology Teachers.

ECO R1 Restriction Enzyme Models
Xerox and cut out one copy for each student.

ECO R1	ECO R1	ECO R1	ECO R1
G A A T T C	G A A T T C	G A A T T C	G A A T T C
C T T A A G	C T T A A G	C T T A A G	C T T A A G

Lab: Macro-DNA Fingerprinting, or Whose Baby is He/She?

Background: It's hard not to turn on the evening news or open a newspaper without one story or article making a reference to DNA "fingerprinting." What are they talking about, does DNA have a distinctive "fingerprint"? Everyone knows that everyone has a distinctive fingerprint on the tips of their fingers, but did you know that everyone's DNA is different as well? Hopefully, you do, but if not, you'll find out how we know and how this idea is used in the real world. In this lab, you are going to simulate using a restriction enzyme to "cut" a piece of your DNA into restriction fragment length polymorphs (RFLP's). We will then separate the pieces we obtain on a simulated gel electrophoresis, which will allow you to distinguish your DNA fingerprint pattern from the others in the class.

But that's not all. It appears that a baby has been found, and the parents are known to be in this class. Using your DNA fingerprint, and the DNA fingerprint of the baby, decide who the "parents" could be.

Objectives:

By using restriction enzymes on a model of a DNA strand, students will be able to see how DNA "fingerprinting" can be used to identify different individuals and related individuals.
Students will use a model of a restriction enzyme, ECO R1, to cut their DNA strand into restriction fragment length polymorphisms (RFLP's).
Students will use these RFLP's to produce DNA fingerprints to determine the parents of a child from the child's own DNA fingerprint.
Students will discuss the bioethical problems concerned with this new technology and the legal problems stemming from it.
Materials (for each person): (for each group of four)
- one set of three paper strips of DNA sequence
- tape
- one ECO R1 Restriction Enzyme Model
- scissors
- one Gel Electrophoresis Sheet
- hole punch

Procedure:

Obtain the three paper strips that represent your DNA sequence. Tape the ends of the paper in increasing number order, being sure to keep the base letters evenly spaced where the strips meet. This is your DNA.
Now take your ECO R1 Restriction Enzyme model. Look at the upper line of code on the model and note the G-A sequence there. The ECO R1 enzyme cuts between the G and the A. Using the hole punch, punch out this G and this A.
Your next step is to cut the DNA sequence into RFLP's. Take your ECO R1 enzyme and place it at one end of the sequence. Move down the sequence, and at each point where there is a G-A sequence appearing in the holes you punched out, cut the DNA sequence between the G and A, taking care not to cut your enzyme (remember, enzymes don't get used up - they are used over and over).
You will end up with several RFLP's of different lengths. Count the number of bases on each of the RFLP's, and place a series of X's in the appropriate boxes on the Gel Electrophoresis Sheet. In an actual electrophoresis gel plate, the largest DNA fragments move the most slowly through the gel, and the smallest DNA fragments move the fastest (and thus the furthest). Thus, they appear as separate lines on the gel electrophoresis sheet.

Questions (answer in COMPLETE sentences):

Compare your DNA sequences to those of your classmates. What differences do you see? What similarities do you see?
Why is the term "DNA fingerprinting" used when we use restriction enzymes to separate DNA into restriction fragment length polymorphisms (RFLP's)?
What effect would using a different restriction enzyme have (one that cuts between, say, a T-T sequence)?
What other uses are there for using DNA fingerprinting?
BIOETHICAL ISSUE - Congress decides that DNA fingerprinting should be done on all newborn infants, for identification purposes and for genetic counseling purposes. Do a little research, and write a short opinion paper on whether this is a good proposal or not. Give specific reasons for your opinions.

Boy's DNA Sequence 1-6

1 G A A T T C G A A T T C G G A A T T C A A G A A T T C A C T G A A
X C T T A A G C T T A A G C C T T A A G T T C T T A A G T G A C T T

1 T T C G A A T T C G A A T T C T G A A T T C T A G A A T T C G A A
Y A A G C T T A A G C T T A A G A C T T A A G A T C T T A A G C T T

1 T T C C G A A T T C G A A T T C T G A A T T C C C G A A T T C T T G
Z A A G G C T T A A G C T T A A G A C T T A A G G G C T T A A G A A C

2 G G A A T T C A G A T G A A T T C A T T G A A T T C A T G A A T T
X C C T T A A G T C T A C T T A A G T A A C T T A A G T A C T T G G

2 C T G A A T T C G G A A T T C G T T C G A A T T C A T C G A A T T
Y G A C T T A A G C C T T A A G C A A G C T T A A G T A G C T T A A

2 T T C C G A A T T C G A A T T C T G A A T T C C C G A A T T C T T G
Z A A G G C T T A A G C T T A A G A C T T A A G G G C T T A A G A A C

3 G G G A A T T C A A G A A T T C A T T G A A T T C A A C C G A A T
X C C C T T A A G T T C T T A A G T A A C T T A A G T T G G C T T A

3 T C A G T C C G A A T T C A T G A A T T C G G T C T G A A T T C T
Y A G T C A G G C T T A A G T A C T T A A G C C A G A C T T A A G A

3 T G A A T T C T G T T G A A T T C G T T T T G A A T T C G G T C C G
Z A C T T A A G A C A A C T T A A G C A A A A C T T A A G C C A G G C

4 G G G G A A T T C G G G A A A G A A T T C T G G A A G A A T T C T
X C C C C T T A A G C C C T T T C T T A A G A C C T T C T T A A G A

4 T T G A A T T C A C C C G A A T T C T T G G G G A A T T C A T T G
Y A A C T T A A G T G G G C T T A A G A A C C C C T T A A G T A A C

4 A A T T C T C G T T T G A A T T C T T T T C G A A T T C G G T C C G
Z T T A A G A G C A A A C T T A A G A A A A G C T T A A G C C A G G C

5 G G T T G A A T T C G A A T G A A T T C A T A A A G A A T T C C A
X C C A A C T T A A G C T T A C T T A A G T A T T T C T T A A G G T

5 A A C T G A A T T C A A A C T C C G A A T T C T C A T T G A A T T
Y T T G A C T T A A G T T T G A G G C T T A A G A G T A A C T T A A

5 C T C T T C C G A A T T C T C G T C C C G A A T T C C T T T G G G C
Z G A G A A G G C T T A A G A G C A G G G C T T A A G G A A A C C C G

6 G A A T T C A A G T T G A A G A A T T C A G G G A A C G A A T T C
X C T T A A G T T C A A C T T C T T A A G T C C C T T G C T T A A G

6 T G A C A T G A A T T C T A T C C G A A T T C T T C A A C T G A A
Y A C T G T A C T T A A G A T A G G C T T A A G A A G T T G A C T T

6 T T C C G G T T T G A A T T C T T T G G T T G A A T T C C T T T T C
Z A A G G C C A A A C T T A A G A A A C C A A C T T A A G G A A A A G

Boy's DNA Sequence 7-9

7 G G A A T T C T G G A A T G A A T T C T T T G C A A G A A T T C A
X C C T T A A G A C C T T A C T T A A G A A A C G T T C T T A A G T

7 G G G A A T C G A A T T C C T T T A A A C C G A A T T C T T T T T
Y C C C T T A G C T T A A G G A A A T T T G G C T T A A G A A A A A

7 T G G C G A A T T C C T T C C C T T G A A T T C T C C G G G C T T T
Z A C C G C T T A A G G A A G G G A A C T T A A G A G G C C C G A A A

8 G G G A A T T C G A A T T C A G A A T T C T T G A A T T C A G A G
X C C C T T A A G C T T A A G T C T T A A G A A C T T A A G T C T C

8 A A T T C T T G A A T T C G A A T T C C G A A T T C T T G G G A A
Y T T A A G A A C T T A A G C T T A A G G C T T A A G A A C C C T T

8 T T C G A A T T C T G A A T T C T T T G A A T T C T T G A A T T C T
Z A A G C T T A A G A C T T A A G A A A C T T A A G A A C T T A A G A

9 G G G G A A T T C A G A A G A A T T C T T C G A A T T C A A G A A
X C C C C T T A A G T C T T C T T A A G A A G C T T A A G T T C T T

9 T T C A G A A T T C T C C G A A T T C C G A A T T C T A A T T T G
Y A A G T C T T A A G A G G C T T A A G G C T T A A G A T T A A A C

9 A A T T C C T C G A A T T C T G A A T T C T C G C G A A T T C T T C
Z T T A A G G A G C T T A A G A C T T A A G A G C G C T T A A G A A G

Girl's DNA Sequence 10-18

10 G G A A T T C A G A A T T C A G A A T T C T T G A A T T C T A T G
X C C T T A A G T C T T A A G T C T T A A G A A C T T A A G A T A C

10 A A T T C T G T A G A A T T C T T G A A T T C T T T G G A A T T C
Y T T A A G A C A T C T T A A G A A C T T A A G A A A C C T T A A G

10 T G A A T T C T T T G A A T T C T T T T G A A T T C G A A T T C C C
Z A C T T A A G A A A C T T A A G A A A A C T T A A G C T T A A G G G

11 C G A A T T C C T G T T G G A A T T C T T G C G A A T T C T G A A
X G C T T A A G G A C A A C C T T A A G A A C G C T T A A G A C T T

11 T T C T T G A A T T C T T G G A A T T C A T T T G A A T T C A G G
Y A A G A A C T T A A G A A C C T T A A G T A A A C T T A A G T C C

11 A A T T C T T A A G A A T T C A G A A T T C T A T A G A A T T C A T
Z T T A A G A A T T C T T A A G T C T T A A G A T A T C T T A A G T A

12 T T G A A T T C T C G A A C G A A T T C T T G A A T T C A A A T A
X A A C T T A A G A G C T T G C T T A A G A A C T T A A G T T T A T

12 G A A T T C T T T G A A T T C T A A A G A A T T C T T G A A T T C
Y C T T A A G A A A C T T A A G A T T T C T T A A G A A C T T A A G

12 T T T T T T G A A T T C A A G A A T T C T T T G A A T T C T T T A A
Z A A A A A A C T T A A G T T C T T A A G A A A C T T A A G A A A T T

Girl's DNA Sequences 13-18

13 T T G A A T T C T G A G A A T T C T T G T G A A T T C A T T G C T
X A A C T T A A G A C T C T T A A G A A C A C T T A A G T A A C G A

13 T G A A T T C T T T C T T G A A T T C T T T G A A T T C T G T C T
Y A C T T A A G A A A G A A C T T A A G A A A C T T A A G A C A G A

13 G A A T T C T A T A G A A T T C T T C C C T T G A A T T C A G A A T
Z C T T A A G A T A T C T T A A G A A G G G A A C T T A A G T C T T A

14 T T G A A T T C T T G T T T T T G A A T T C A A G T T T T G A A T
X A A C T T A A G A A C A A A A A C T T A A G T T C A A A A C T T A

14 T C A T G T A A G A A T T C T T G G T G A A T T C A T T T G A A T
Y A G T A C A T T C T T A A G A A C C A C T T A A G T A A A C T T A

14 T C A T T T A T G A A T T C G A A A A T T G A A T T C T T A C C T T
Z A G T A A A T A C T T A A G C T T T T A A C T T A A G A A T G G A A

15 T T T T T G A A T T C T T A G T G A A T T C C C C T G T G A A T T
X A A A A A C T T A A G A A T C A C T T A A G G G G A C A C T T A A

15 C T G C C C T T A A G A A T T C T T T G T T T G A A T T C A T G G
Y G A C G G G A A T T C T T A A G A A A C A A A C T T A A G T A C C

15 G T A T G A A T T C T G G G A T T A T G A A T T C T A T A T A G G T
Z C A T A C T T A A G A C C C T A A T A C T T A A G A T A T A T C C A

16 T T G G A A T T C G A A T T C C T T A G A A T T C A G A A T T C A
X A A C C T T A A G C T T A A G G A A T C T T A A G T C T T A A G T

16 T A G A A T T C A T G A A T T C A T A G G A A T T C T A T G A A T
Y A T C T T A A G T A C T T A A G T A T C C T T A A G A T A C T T A

16 T C G A A T T C A G A A T T C A A A G A A T T C G A A T T C T T A T
Z A G C T T A A G T C T T A A G T T T C T T A A G C T T A A G A A T A

17 T T T G A A T T C G T T T A G A A T T C T G A A T T C T T G A A T
X A A A C T T A A G C A A A T C T T A A G A C T T A A G A A C T T A

17 T C T T A C G A A T T C T T T G A A T T C A G A A T T C A A A T T
Y A G A A T G C T T A A G A A A C T T A A G T C T T A A G T T T A A

17 G A A T T C A A A T G A A T T C T T G A A T T C A G A A T T C T A T
Z C T T A A G T T T A C T T A A G A A C T T A A G T C T T A A G A T A

18 A G A A T T C G A A T T C A A G A A T T C G G A G G A A T T C T T
X T C T T A A G C T T A A G T T C T T A A G C C T C C T T A A G A A

18 T G A A G A A T T C G A A T G A A T T C T G G A A T T C C A A G G
Y A C T T C T T A A C C T T A C T T A A G A C C T T A A G G T T C C

18 A A T T C A A T A G A G A A T T C A A T T G A A T T C A T T C T T T
Z T T A A G T T A T C T C T T A A G T T A A C T T A A G T A A G A A A

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Macro-DNA: Doing DNA Fingerprinting and Gel Electrophoresis on a Large Scale

Teacher Notes

References:

Lab: Macro-DNA Fingerprinting, or Whose Baby is He/She?

Macro-DNA: Doing DNA Fingerprinting
and Gel Electrophoresis on a Large Scale