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.
6. Repeat if necessary

Note: Prepare enough buffer solution to allow each group to cover the gel in the chamber to a depth of about 2 cm. Depending on the type of electrophoresis units being used, the amount of buffer needed could be as much as 300 mL per chamber. The gel preparation requires an additional 60 mL of buffer to make a 6 x 6 cm gel.

Make fresh buffer weekly.

Safety Precautions

Be sure all connecting wires, terminals and work surfaces are dry before using the electrophoresis units. Electrical Hazard: Treat these units like any other electrical source—very carefully! Do not try to open the lid of the unit while the power is on. Wearing chemical splash goggles and gloves is strongly recommended. Wash hands thoroughly with soap and water before leaving the laboratory. Please consult 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 solutions used in this lab may be disposed of down the drain using copious amounts of water according to Flinn Suggested Disposal Method #26b. Used 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 sucrose, which may be disposed of by Flinn Suggested Disposal Method #26b.

Lab Hints

• Enough materials are provided in this kit for 30 students working in groups of five or for six groups of students. Both parts of 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 50 minutes. Gel preparation requires approximately 10–20 minutes plus at least an additional 20 minutes for the gel to solidify (60 minutes is optimal solidification time). Longer solidification times “harden” the gel, minimizing tears and creating more distinctive bands of DNA.
• The gel preparation pages have been listed separately so that they may be copied for student use if desired.
• All of the simulated DNA used in this activity are negatively charged and will all run in the positive direction. Therefore, the combs do not need to be placed in the center of the gel and may be placed towards 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 agarose solution.
• Without a balance, prepare all six 0.8% gels simultaneously. Measure 375 mL of electrophoresis buffer in a 500-mL Erlenmeyer flask, add the entire 3 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 solution may be stored at room temperature.
• Sucrose and/or glycerin have been added to the simulated DNA samples to make them denser than the TAE buffer. This causes the sample to sink into the sample well in the gel.
• TBE buffer may be used in place of the TAE buffer included with this laboratory kit.
• A 10-µL micropipet with disposable tips may be used instead of the disposable needle-tip pipets.
• Any empty wells should be end wells since they tend to run the quickest.
• Bands should be visible after 15–20 minutes at 125 V.
• Remind students that if a band is present at all, even if it is faint or touching another band, the sample is positive for the “trait.”
• Gels may be reused. Allow the used gel to soak in TAE buffer for several hours or overnight and the dye bands will disappear and the gel may be reused. Due to this, 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 number 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.

• For more advances classes, have students complete a research project or paper on genetics of human eye color, hair color or genetic counseling.

Answers to Prelab Questions

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

2. Decode the bands for each parent and child using Figure 1 in the Background section. What traits does each person possess?

Father—Brown hair, blue eyes, no freckles
Mother—Brown hair, brown eyes, freckles
Child 1—Brown hair, brown eyes, no freckles
Child 2—Blond hair, blue eyes, freckles
Child 3—Brown hair, blue eyes, freckles

3. Were any traits the children had that the parents did not have? What does this tell us about the genotypes of the parents?

Both parents have a phenotype of brown hair and we see that child 2 has blond hair. This shows that both parents must have a heterozygous genotype, that is, both parents carry blond alleles although they are not expressed. Each child has a 25% probability of having a blond phenotype with heterozygous parents. This may be demonstrated using a Punnett Square.

4. Using terms from this activity, explain why people look different even when closely related.

Accept all reasonable answers that include at least a couple of terms from the background sections (e.g., genotype, phenotype, heterozygous, homozygous, genes, dominant, recessive).

5. Genetic counseling is an up and coming branch of medicine that involves the use of tests, such as gel electrophoresis, to find out more information on the genotypes of a couple to identify any recessive diseases that may potentially affect their children. A couple discovers through genetic counseling that both of them are carriers of a mutated CFTR gene characteristic of the genetic disease cystic fibrosis. Although neither of them have the disease, what are the chances of their first child having the disease if it is a recessive disease? Draw a Punnett Square if needed.

Cystic fibrosis is a recessive disease, meaning two copies of the allele must be inherited for the disease to be prevalent. Every child of a heterozygous couple has a 25% chance of having the disease.

Teacher Handouts

10784_Teacher1.pdf

Introduction

Every person has a characteristic DNA fingerprint—3.2 million base pairs long, containing more than 30,000 genes. No two people, except identical twins, have the same DNA sequence. Variations in DNA fingerprints arise as DNA from two genetically different people combine during reproduction. Knowing that children inherit genes from both parents explains why children often resemble one or both of their parents due to common traits. How then can we genetically explain a child who is born with a trait neither parent has?

Concepts

• DNA fingerprinting

• Biotechnology
• Gel electrophoresis
• Genes

Background

Each individual carries two copies of each gene called alleles. One copy is inherited from the mother and the other from the father. An individual can thus have two copies or forms of a gene that are identical, or they may have two different alleles.

A genotype is the information encoded on a gene in DNA. Genotype cannot be seen and may only be determined through laboratory tests, such as gel electrophoresis. A phenotype is the trait that may be physically observed from the outside. Homozygous (homo = same) refers to a gene in which both alleles are of the same type, which may be either dominant or recessive. Heterozygous (hetero = different) is the term used to describe a gene which contains one dominant allele and one recessive allele. If we observe a phenotype representing a dominant trait, we are unable to determine if the genotype is in fact homozygous dominant or heterozygous. However, when we observe a recessive phenotype, it is always indicative of a double homozygous recessive genotype since two copies of the recessive allele must be present for the trait to be observable. On paper, alleles forms are expressed as two letter series with dominant alleles represented by capital letters and recessive alleles with lower case letters. Crosses between two individuals using a Punnett Square may be used to find the probability offspring’s genotypes for a specific gene. However, certain traits are more complex and are controlled by more than one gene. Human eye color is determined by at least three genes and each gene has two alleles. Only two out of these three genes, the brown-blue gene and a green-blue gene, are currently well understood by scientists.

We observe genetics in action on a daily basis. For example, maybe you or one of your siblings possesses a trait neither of your parents has. Or maybe you know someone whose parents both have brown hair yet the child is blond. Examples such as these would indicate that both parents must be heterozygous for the particular trait. This means that they carry the recessive trait although it is not expressed in their own phenotype. Since human eye and hair color inheritance are somewhat complicated involving more than one gene, a simplified “brown hair is dominant to blond hair” and “brown eyes are dominant to blue eyes” will be used for this activity.

DNA fingerprinting methods were first published in 1984 by Sir Alec Jeffries. Sir Jeffries studied inherited variations within genes to determine the cause of specific genetic diseases. Sir Jeffries discovered that certain enzymes cut the DNA sequence at specific points. When these samples were analyzed using gel electrophoresis, a unique banding pattern was produced. Unique DNA fingerprints result from variations in DNA created by mutation and crossover during parental meiosis. Specifically, variations in DNA sequences are created by changes in the number of repeating DNA base-pair sequences between genes on the chromosomes. Every living organism produced by sexual reproduction (except identical twins) has a unique number of tandem DNA repeats called variable number of tandem repeats (VNTR). Since these tandem repeats are not part of a gene, they do not affect the viability of the organism. Sir Jeffries’ methods have been slightly modified and refined for use in DNA sequencing laboratories throughout the world today.

In gel electrophoresis, DNA test samples are separated by exposure to an electrical current that flows from negative to positive (cathode to anode). 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 may then be required in order to view DNA fragment bands since they are colorless in the gel. Other substances, such as organic dyes, may be visible immediately without the use of a stain.

In the media, we often hear DNA testing being used in criminal investigations. These tests include running gel electrophoresis and comparing the banding pattern of known standards of human DNA against the crime scene DNA and the suspect’s DNA. A match is determined by calculating the probability of an individual having a particular combination of bands in a population. The more matching bands that are observed in the electrophoresis gel, the more likely the DNA comes from the same person.

A second sample may be run for confirmation using a different restriction enzyme. A different banding pattern will be revealed for the same DNA samples because the DNA sequence is cleaved at a different base-pair location. The odds that two different individuals (again with the exception of identical twins) will have identical banding patterns for two different DNA cuts tends to be greater than the current human population. The theoretical risk of a coincidental match is 1 in 100 billion.

As always, good scientific protocol is critical to the outcome of any laboratory analysis. Sloppy work might convict the wrong person or let a guilty suspect go free. Consequently, analysts must carefully document which restriction enzyme was used, the conditions and chemicals that were used, and the names of all known standards and controls that were prepared with the DNA sample.

In this experiment, the bands observed in the gel after electrophoresis will be several different colors. Each color will represent a particular phenotype for a specific trait (see Figure 1).

It is important to remember that this activity is a simulation of the type of an analysis that is made using DNA fingerprinting. In this activity, only the phenotypes of the unknown individuals will be discovered after running the gel—only the gene expression will become visible. In “real” DNA gel electrophoresis, the genotypes (allele expression) of the individuals would be visible. DNA gel electrophoresis would thus reveal if the genes are heterozygous (two different alleles are found including one that is not visible by physical observation) or homozygous (both alleles are identical copies of either the dominant or recessive form) for each trait. In addition, if real DNA samples were used the gels would need to be stained in order to view the bands in the gel. Since simulated DNA is used in this lab, the dying step is not necessary. Colorful bands will be visible after the gel has run for 15–20 minutes, and results may be read the same lab period.

Experiment Overview

In this activity simulated DNA will be analyzed using gel electrophoresis to take a deeper look into the inheritance pattern of a family’s traits. Nothing is known about the phenotypes of the “subjects” prior to running the gel. Therefore, the gel must be run properly to ensure that the correct results are obtained. The genotypes of the parents can be inferred once the phenotypes of their three children are viewed.

Materials

Safety Precautions

Be sure all connecting wires, terminals and work surfaces are dry before using the electrophoresis units. Electrical Hazard: Treat these units like any other electrical source—very carefully! Do not try to open the lid of the unit 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 apron. Wash hands thoroughly with soap and water before leaving the laboratory.

Procedure

Part A. Loading a Gel

1. Depending on the type of electrophoresis unit used, assemble the 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 a 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.
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 2).

8. Shake the simulated DNA sample tube well and allow it to settle. After a few seconds, withdraw 10 μL of Simulated DNA—Father 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 3).

9. Dispense the sample into the first well 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 4).

10. Use 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.)

Simulated DNA—Mother, Well 2
Simulated DNA—Child 1, Well 3
Simulated DNA—Child 2, Well 4
Simulated DNA—Child 3, Well 5

11. Well 6 may be left empty.

Part B. Running a Gel

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

10784_Student1.pdf