Materials Included In Kit

Additional Materials Required

Prelab Preparation

Preparation of 1X Electrophoresis Buffer

1. Measure 20 mL of 50X TAE buffer in a graduated cylinder.
2. Add the 50X buffer to 980 mL of distilled water in a 1000-mL Erlenmeyer flask.
3. Mix with a glass stirring rod.
4. Seal with Parafilm^® or plastic wrap.
5. Label and store in a refrigerator. Note: Allow buffer to return to room temperature before use.
6. Repeat if necessary.

Safety Precautions

Be sure all connecting wires, terminals and work surfaces are dry before using the electrophoresis unit. Electrical Hazard: Treat this chamber like any other electrical source—very carefully! Do not try to open the lid of the unit while the power is on. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Remind students to wash their hands thoroughly with soap and water before leaving the laboratory. Please review current Safety Data Sheets for additional safety, handling and disposal information.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. All leftover solutions used in this lab may be disposed of down the drain with plenty of excess water according to Flinn Suggested Disposal Method #26b. Agarose gels may be disposed of in the regular trash according to Flinn Suggested Disposal Method #26a. The “DNA” in this kit is simulated—it does not contain any real DNA products. Each sample contains a mixture of dye solutions and glycerin, which may be disposed of by Flinn Suggested Disposal Method #26b.

Lab Hints

• Enough materials are provided in this kit for 20 gels. This laboratory activity can reasonably be completed in one 50-minute class period if the gels are prepared and the electrophoresis equipment is ready for use. Electrophoresis setup, sample transfer and the start of electrophoresis will take approximately 20 minutes.
• Reuse TAE buffer in the electrophoresis unit for up to one week.
• The gel preparation pages have been listed separately so they may be copied for student use if desired. Gel preparation requires approximately 10–20 minutes plus at least an additional 20 minutes for the gel to solidify (60 minutes is optimal). Longer solidification times “harden” the gel, minimizing tears and creating more distinctive bands of DNA.
• All of the simulated DNA samples used in this activity are negatively charged and will therefore migrate toward the positive electrode. The combs for forming the wells should be placed at one end of the gel, rather than in the center, and the gel is then oriented in the electrophoresis chamber with the wells near the cathode.
• Teachers who are familiar with gel electrophoresis using DNA or protein samples may be surprised by the appearance of the dye bands. Dyes bands are wider and less defined than bands typically observed when DNA or proteins are run in a gel.
• It may be a good idea to introduce students to the basic principles of electrophoresis and genetics prior to this lab activity.
• When preparing agarose solution using a stirring hot plate, rotate a magnetic stir bar very slowly to diminish the number of bubbles in the solution.
• If a balance is not available, prepare all 20 0.8% gels simultaneously. Measure 1250 mL of TAE electrophoresis buffer in a 2-L Erlenmeyer flask, add the entire 10 g of agarose, and dissolve as directed.
• It is a good idea to keep an extra prepared gel on hand to cover Murphy’s Law.
• The simulated DNA samples, concentrated TAE buffer, and agarose may be stored at room temperature.
• Glycerin has been added to the simulated DNA samples to make them more dense than the TAE buffer. This causes the samples to sink into the sample wells in the gel.
• A 10 µ L micropipet with disposable tips may be used instead of the disposable needletip pipets provided in this kit.
• Separate bands should be visible after 15–20 minutes at 125 volts.
• Remind students that if a band is present at all, even if it is faint or touching another band, the sample contains that STR band.
• Gels may be reused—allowing the used gel to soak in TAE buffer for several hours or overnight will cause the dye bands to diffuse out and disappear. Because the dyes diffuse into the gel buffer, students must read the gels the same day they are run. Gels may not be saved overnight and read the following day as the bands will no longer be present.

Teacher Tips

• Have students practice pipetting before performing this lab. The Pipetting Practice Kit, Flinn Catalog No. FB1649, is a reusable, durable option that works very well. Alternatively, students may pipet 10 μL of colored water into a defective gel, or a practice gel may be made with inexpensive agar rather than agarose.

Answers to Prelab Questions

1. Archeologists would like to determine which mummies are related to King Tutankhamen. Which DNA fingerprinting technique would be the most useful to determine the male relatives? Why?

   Y-chromosome analysis is the most useful technique to determine male relatives because it is the only chromosome definitely passed on from one male to his male descendents.

2. Why is it important to match more than one STR to determine relatedness?

   The possibility of multiple STRs matching in non-related individuals decreases with the number of STRs matched.

Sample Data

1. Draw the bands observed in the gel. If available, use colored pencils. Otherwise label the bands with the color observed.
   {11104_Data_Figure_3}

Answers to Questions

Post-Lab Analysis

2. Decode the bands for each sample by filling in the table above. Who is most likely related to John?

   John is most likely related to the Chevalier family since they both have the FGA and TPOX STR sequences but not the D7S820, D15S51 or D21S11 STR sequences.

Teacher Handouts

11104_Teacher1.pdf

References

Human Genome Project Information http://www.ornl.gov/sci/techresources/Human_Genome/home.shtml (accessed March 2011).

Orchid Cellmark http://www.orchidcellmark.com/forensicdna/mtdnaanalysis.html (accessed March 2011).

Introduction

Each person has a characteristic DNA fingerprint arising from its unique sequence of 3.2 billion base pairs and containing 20,000 to 25,000 genes. No two people have the same DNA sequence. Variations in DNA fingerprints occurs because DNA from two genetically different people combine during reproduction. Since a child inherits genes from both parents, DNA can be used to determine relatedness.

Concepts

• Biotechnology
• Electrophoresis
• DNA fingerprinting
• Relatedness testing

Background

After paying $1 for an old downtown mansion that needed to be moved or it would be torn down, the contractor discovered a hidden treasure concealed in a false wall in the basement. The Queen Anne–style house was built in 1890. It is being moved to make way for a wider road through town. As the contractor prepared the home for transport to its new location, some of the basement wall was removed to more easily stabilize the structure. Contained within the small hidden space was a Mason jar. The jar contained a lock of hair labeled “John’s first haircut” and two baby teeth wrapped in fabric. A second piece of fabric contained $10,000. The contractor wanted to reunite the money and family mementos with the correct family. According to county records, the house had changed hands five times before the city acquired it. Descendents of all five former owners willingly supplied DNA samples to compare to DNA extracted from the baby teeth.

DNA profiling or “fingerprinting” is an accurate way to match DNA to the original source or to a relative, especially a parent or child as well as grandchildren. Special enzymes called restriction enzymes cleave long strands of DNA between specific sequences of base pairs. Different restriction enzymes cleave the DNA at different locations, with each enzyme recognizing a characteristic sequence of base pairs where it will cut a DNA molecule. Since the DNA found in your cheek cells is the same as that found in your white blood cells, the fragments created by treating both DNA samples with the same restriction enzyme will be identical. A child inherits half of his or her genetic code from each parent. Therefore the fragments created from treating a child’s DNA will be a mixture of those found from the two parents.

The entire strand of DNA is not usually sequenced in DNA fingerprinting. Instead, specific sections of DNA are analyzed. Typically, these are short sections of a repeating series of DNA called short tandem repeats (STR). Contained within each strand of DNA are coding and non-coding regions. The coding regions are the genes that code for specific proteins that do much of the work in the cell. Non-coding regions often consist of repeats of the same base sequence. STR repeats are 2–6 base pairs long. In related people, the number of times an STR repeats in one area on a single chromosome is likely to be the same. These regions are targeted for analysis. The more STR sequences that are analyzed, the more significant the match that can be made in DNA forensics. In the United States a specific set of thirteen STR sequences is usually analyzed.

Two special cases of relatedness testing are used—Y chromosome testing and mitochondrial DNA testing. Since the entire Y chromosome is passed on from the father to a son, the Y chromosome is the ideal chromosome for determining if one male is directly related to another man. A grandfather, father, and son all share the same STR pattern on the Y chromosome. Mitochondria are inherited solely from the mother. A mother’s mitochondrial DNA can be compared with that of her children and those of her daughter’s children, etc. through the maternal line. Thousands of mitochondria are found in a single cell whereas there is only a single set of nuclear DNA. Also, nuclear DNA degrades more quickly over time. Thus mitochondrial DNA sequencing is often used with highly degraded or extremely small samples of DNA. Mitochondrial DNA analysis is used on hair and teeth samples that do not have the root attached, such as the sample of baby’s first haircut or baby teeth that have fallen out.

DNA fragments obtained using restriction enzymes are separated using gel electrophoresis by exposure to an electrical current. After the sample has migrated through the gel and DNA fragments have separated, the gel is carefully removed from the electrophoresis chamber. Use of a stain is required in order to view the bands arising from DNA fragments since they are colorless in the gel. Other substances, such as organic indicator dyes, may be used in simulations as the dyes are visible immediately without the use of a stain.

In this experiment, the bands observed in the gel after electrophoresis will be several different colored dyes. Each color represents a particular STR, as shown in Table 1.

Experiment Overview

In this activity, six simulated DNA samples will be analyzed using gel electrophoresis to determine which family described in the background scenario is entitled to receive the mementos and money.

Materials

Prelab Questions

1. Archeologists would like to determine which mummies are related to King Tutankhamen. Which DNA fingerprinting technique would be the most useful to determine the male relatives? Why?
2. Why is it important to match more than one STR to determine relatedness?

Safety Precautions

Be sure all connecting wires, terminals and work surfaces are dry before using the electrophoresis unit. Electrical Hazard: Treat this chamber like any other electrical source—very carefully! Do not try to open the lid of the chamber while the power is on. Use heat protective gloves and eye protection when handling hot liquids. Dyes will stain skin and clothing—avoid all contact. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory. Please follow all laboratory safety guidelines.

Procedure

Part A. Loading the Gel

1. Assemble the electrophoresis unit according to the teacher’s instructions.
2. Place the electrophoresis unit in a horizontal position on top of a piece of white paper on a level table or countertop. Note: Do not move the unit after loading the samples.
3. Gently slide an agarose gel from a zipper-lock bag into the casting tray with the wells adjacent to the cathode (–) end of the unit.
4. Carefully position the gel and tray into the electrophoresis chamber. Caution: Be careful not to break or crack the gel. If the gel is damaged it cannot be used. Promptly inform your instructor of any damaged gels.
5. Obtain approximately 150 mL of electrophoresis buffer in a 250-mL Erlenmeyer flask.
6. Pour enough electrophoresis buffer into the unit to submerge the entire gel surface to a depth of 2–3 mm. If the gel begins to float away, reposition it on the tray.
7. By convention, gels are read from left to right (see Figure 1).
   {11104_Procedure_Figure_1}
8. Shake the simulated DNA sample tube well and allow the liquid to settle. After a few seconds, withdraw 10 μL of Simulated DNA—Chevalier family sample, by filling only the needle tip of a clean pipet. Note: Fill the tip by squeezing the pipet just above the tip, not the bulb. Be careful not to draw the sample further up the pipet (see Figure 2).
   {11104_Procedure_Figure_2}
9. Dispense the sample into Well 1 by holding the pipet tip just inside the well. The sample will sink to the bottom of the well. Caution: Do not puncture the bottom or sides of the well. Do not draw liquid back into the pipet after dispensing the sample (see Figure 2).
10. Using a fresh pipet for each sample, repeat steps 8 and 9 for the remaining Simulated DNA Samples as follows: (Give each group member a chance to add a sample.) Gonzalez family sample—Well 2 John’s sample—Well 3 Li’ family sample—Well 4 O’Connor family sample—Well 5 Sarr family sample—Well 6

1. Place the lid on the electrophoresis chamber and connect the unit to the power supply according to your teacher’s instructions.
2. Run the gel as directed by your teacher at 125 volts for 15–20 minutes. Note: Bubbles will be observed along the electrodes in the chamber while the sample is running. The bubbles are the result of the electrolytic decomposition of water— hydrogen is generated at the cathode and oxygen is produced at the anode.
3. Turn off the apparatus to stop the gel according to your teacher’s instructions.
4. When the power is off, remove the cover and carefully remove the gel tray from the chamber. Place the gel tray on a piece of paper towel or light box. Note: Be careful not to break or crack the gel.
5. Consult your instructor for appropriate disposal procedures.

Student Worksheet PDF

11104_Student.pdf