Materials Included In Kit

Additional Materials Required

Prelab Preparation

Enough copies of the DNA Sequencing Worksheet should be made for classroom use.

Safety Precautions

This simulation activity is considered safe but follow all normal laboratory safety rules. Provide usual precautions for using scissors.

Disposal

Materials from this activity may be stored for future use.

Teacher Tips

• Enough materials are provided in this kit for 30 students working in pairs or for 15 groups of students. The laboratory can be completed in one 50-minute class period.

• This activity assumes a working knowledge of DNA structure and function as well as gel electrophoresis. The concept in this activity is not an easy one but the manipulative materials will help students visualize the procedures.
• The materials provided in this kit can be used as a guided discussion as well as an individual laboratory activity with all student groups doing the steps simultaneously with teacher direction.

Sample Data

Part I.

Record fragment lengths possible for each nucleotide type.

Part II.

Draw a line for each fragment length for each nucleotide base type on the simulated gel electrophoresis.

Write the sequence for the base pairs from this simulated electrophoresis gel.

Part III.

Write the sequence of bases shown by the following electrophoresis results.

Teacher Handouts

10453_Teacher1.pdf

References

The Dynamics of DNA Sequencing, Morvillo, N. The Science Teacher, 1997, 64, 46–50.

Introduction

Remember when the newspaper headline read: “Sequencing of the Human Genome Complete.” There are over 3,000 million base pairs in the human genome sequence! How did biologists determine the sequence of all those base pairs?

Concepts

• Sanger sequencing method

• Dideoxynucleotides
• Electrophoresis
• Nucleotides
• Phosphodiester bonds

Background

When historians sort through all the noteworthy developments of the 20th century, two accomplishments are likely to surface as the most significant. One was the advent of the computer chip. The influence of the computer in our everyday life is impossible to avoid. The second development might ultimately have even more impact on the human race, namely, the deciphering of the human genome. Determining the sequence of the base pairs in human DNA and creating a “library” of the entire human genome will have influence on science and medicine for generations to come. All 3,000,000,000 base pairs in the 30,000–35,000 genes have been sequenced. Key disease-causing genes are already being researched.

The type of DNA sequencing done today is based upon a method devised by Frederick Sanger in 1977 called the Sanger-dideoxysequencing method or the Sanger chain-termination method. The technique is based upon copying a DNA molecule and then terminating the replication process at a specific nucleotide. (This activity assumes a working knowledge of basic DNA structure, DNA replication and gel electrophoresis. If these basic ideas are not known, consult basic biology textbooks to review these ideas before proceeding.)

The key building blocks for nucleic acids are the nucleotides. A generic RNA nucleotide, for example, is illustrated in Figure 1.

A generic DNA nucleotide is shown in Figure 2.

Note that the main difference between an RNA nucleotide and a DNA nucleotide is that there is an OH group on both the 3′ and 2′ carbons of the ribose sugar in RNA but in the DNA nucleotide there is an OH group on only the 3′ carbon. There is an “O” missing on the 2' carbon. Thus, the name deoxyribose and deoxyribonucleic acid.

As nucleotides are joined together to form a DNA chain, the phosphate group from the incoming nucleotide attaches to the 3′ OH group of the sugar in the last nucleotide of the growing chain. A bond is formed at this location and is called a phosphodiester bond. This is shown in Figure 3.

The key to understanding the Sanger chain-termination method is the use of dideoxyribose in a DNA nucleotide. A dideoxynucleotide is diagrammed in Figure 4.

Notice that there is not an OH group on the 3′ carbon. This is significant because a phosphodiester bond cannot form between a phosphate and a 3′ OH group on this nucleotide (since the nucleotide has no OH groups). If a dideoxynucleotide gets into a growing nucleotide chain, it has the effect of terminating further growth of the chain because it doesn’t have the 3′ OH group for the next phosphodiester bond to form in the chain. The chain will not continue to grow in length because there is no place for another nucleotide to attach.

In a test tube, the Sanger chain-termination method makes copies of the DNA in a process similar to how a cell replicates DNA. The first step in the replication process is the separation of the two complementary strands. In the cell, enzymes are involved. In vitro, heat (95 °C) is used to achieve this separation. After applying heat to separate the strands, the temperature is lowered to allow the replication to proceed. When the new, complementary strand has been synthesized, the DNA molecule is double stranded. If the temperature is raised again, the strands will separate and another replication can be initiated. If this process of heating and cooling is repeated over and over, many copies of the DNA molecule or sequence can be generated.

For a sequencing project, the replications are done in four separate test tubes. Each tube contains the following items:

1. DNA polymerase—the enzyme which catalyzes the elongation of new DNA at the replication fork. This enzyme joins nucleotides together via phorphodiester bonds. Taq DNA polymerase is used since it can withstand high temperatures.
2. Primer—a short, single-stranded piece of DNA that binds to the DNA template to signal the polymerase where to start copying.
3. The template DNA.
4. Deoxynucleotides—the four nucleotides (A, T, C, G) are added to each tube. Then each tube is given a different key ingredient—a small amount of one of the dideoxynucleotides. One tube gets dideoxynucleotides containing adenine, another gets a dideoxynucleotide with thymine, another with cytosine and the fourth with guanine. The dideoxynucleotides are labeled with a radioactive isotope so that they can be detected later. This also allows the readings to be done by an isotope-reading computer. (Machines that perform all of these operations have been key to sequencing the human genome.)

When each tube is heated and cooled, many replicated sequences are created. Because the dideoxynucleotides are in the test tube, random additions of the dideoxynucleotides will result in DNA segments of various lengths (since a chain is stopped after the addition of one of the dideoxynucleotides). In the tube with the adenine dideoxynucleotide, there will be segments of various lengths that were terminated wherever there was an adenine in the original double-stranded DNA. Each length of fragment indicates a location of an adenine base in the original DNA molecule.

The fragments generated in the test tube are then separated by electrophoresis to determine the lengths of the generated fragments. This is done for all four bases (A, T, C, G) and the four electrophoresis gels are then analyzed to determine the original sequence in the DNA molecule. This entire process can be simulated in this activity.

Materials

Safety Precautions

This simulation activity is considered non-hazardous. Follow all standard laboratory safety guidelines.

Procedure

1. Secure one each of the DNA Sequencing Cutout Sheets 1–4.
2. Cut all of the sheets carefully along the dotted lines.
3. Note the three DNA template strips (the long strips with shaded sugars). Tape the three DNA template strips together matching the phosphate tab #1 with the OH #1 and the phosphate tab #2 with the OH #2. When the DNA template is assembled, the base pairs from top to bottom should be in the following order: G–A–G–T–C–A–C–T–C–T–A–A–G–T–A.
4. Place the template on a lab table where it is clearly visible.
5. Separate the dideoxynucleotides from the regular deoxynucleotides and set them aside.
6. Separate the deoxynucleotides into separate piles, one for each type (A, T, C, G).
7. Perform your test tube simulations in the order A, T, G and C. For each test tube simulation, complete steps 8–15.
8. Locate the adenine dideoxynucleotide and place it in the pile with the adenine deoxynucleotides. Shuffle it into the pile so that its location is not obvious when drawing one from the pile.
9. Locate the DNA primer fragment. Determine where it will match and join the DNA template strand. Remember the 3′–5′/5′–3′ opposite orientation. Place the primer fragment in place on the template DNA.
10. Start to build a complementary strand along the DNA template. Draw the appropriate nucleotides from the nucleotide piles as needed to complete the complementary strand. Place them in the growing chain on the table top. (Remember, adenine pairs with thymine and cytosine with guanine.)
11. When the dideoxynucleotide is drawn from the pile, place it in the new strand. This addition prevents the new DNA strand from growing further since it does not contain the 3′—OH group needed to form the next bond with an additional nucleotide. Note what happens when an attempt is made to add another nucleotide to the model.
12. Count the number of base pairs in the DNA chain fragment that has been created. Record this number on Part I of the DNA Sequencing Worksheet for adenine. (Do not include the primer as a part of the fragment.)
13. Remove the entire fragment—except the primer—and return the nucleotides to their appropriate piles. Note: Removing the fragment and starting over again simulates the process occurring in the test tube during the heating/cooling process. In the test tube, however, all of the fragments would stay whole and be analyzed at the end of the experiment.
14. Start forming another new complementary strand as before and stop when the addition of an adenine dideoxynucleotide terminates the chain building. Record the length of the new DNA fragment.
15. Continue to make strands until strands have been made that terminate at all of the adenine locations on the template. Record the length of each strand. Note: Due to the random nature of the activity this may take more trials than the number of different length fragments.
16. Repeat steps 8–15 three more times using the thymine, guanine, and cystosine dideoxynucleotides in turn. Record all of the fragment lengths in the appropriate place on Part I of the DNA Sequencing Worksheet.
17. Complete Part II of the DNA Sequencing Worksheet.
18. Complete Part III of the DNA Sequencing Worksheet.
19. Consult your instructor for appropriate disposal procedures.

Student Worksheet PDF

10453_Student1.pdf