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DNA Sequencing Simulation
(Sanger Method)

Regina Lindsey-Lynch
1994 Woodrow Wilson Biology Institute


Student Information

Background Information: The Sanger sequencing method, which is also known as the dideoxychain termination method, produces a nested set of radioactive fragments from a template strand by replicating the template strand to be sequenced and interrupting the replication at one of the four bases.

To start the process, the DNA segment of interest is heated to disassociate the strands. Then four separate reaction mixtures are set up. Within each reaction mixture are the following items: a DNA segment of unknown sequence, DNA polymerase, a primer, a mixture of four naturally occurring nucleotides (of which some of the adenines are radioactive), and one of the four dideoxynucleotides. A dideoxynucleotide cannot bond to other nucleotides because its 3' end is modified.

A primer template, which is a short known sequence of DNA, is attached to the 3' end of the single strand of DNA. The primer is essential to initiate the replication of the templates by DNA polymerase. The DNA polymerase catalyzes the synthesis of a complementary strand of DNA. When dideoxynucleotides are incorporated, strand synthesis stops, and shorter segments result. Eventually, strands of DNA of all varying lengths will have been randomly synthesized. These strands are then put into wells in a polyacrylamide gel, and an electric current is run through the gel. The synthesized DNA segments are sorted by size: short ones travel further than long ones. An autoradiogram is made of the gel, and the DNA sequence is visualized and interpreted.

Objective:
This lab will simulate the Sanger method of sequencing DNA, both visually and kinesthetically. It should be used in conjunction with a reading on Sanger sequencing. This simulation uses colored pop-beads, which represent nucleotides and dideoxynucleotides. The dideoxynucleotides have their "stubs" cut off. These are denoted by an * in the materials section.

Materials:
A. Reaction mixture bags
These should consist of four large baggies, each containing 4 smaller baggies of pop-beads. The color code for the nucleotides is below. A red dot should be painted on half of the plain adenines of each reaction mixture. This denotes that these are radioactive.

  • blue = adenine
  • green = thymine
  • yellow = guanine
  • orange = cytosine

The "mixtures" should be counted out as follows:

Reaction mixture 1: Reaction mixture 2:
50 adenine, 25 dideoxyadenine*75 adenine
75 thymine*50 thymine, 25 dideoxythymine*
75 guanine*75 guanine
75 cytosine* 75 cytosine
Reaction mixture 3:Reaction mixture 4:
75 adenine75 adenine
75 thymine75 thymine
50 guanine, 25 dideoxyguanine*75 guanine
75 cytosine50 cytosine, 25 dideoxycytosine*

B. Masking tape
The masking tape is used to tape off a table top in the outline of an electrophoresis chamber with four wells, labeled A,C,G, and T respectively, to run your reaction mixtures.

Procedure:

  1. Fill in the open half of the DNA strand (figure 1) with the appropriate nucleotide letter. Check that these are correct, as these will form the groups of nucleotides from the single stranded side of the DNA in your DNA sequencing.

  2. Obtain a "reaction mixture" bag of nucleotide/dideoxynucleotide pop-beads. There are four different bags (see materials section). Each lab group will prepare sequences from only one "reaction mixture." Class results will be combined in the "giant electrophoresis chamber," which is taped on the front table.

  3. As practice, assemble the first strand from your "reaction mixture" bag as a complete complementary strand to the single strand of DNA in figure 1. Starting with the 5' end of a green pop-bead, build the chain from right to left. Do not use any dideoxy pop-beads in this strand. The primer/primer template is a constant in the sequencing reaction, so just ignore it.

  4. Start selecting nucleotides to build more DNA sequences. Try not to look into your nucleotide bags as you select. Once again, construct your sequences from right to left. When a dideoxynucleotide is added, no more plain nucleotides can be added. (It is physically impossible!!) You will end up with a variety of different segment lengths of DNA. Continue until you have 10-12 DNA segments, or until you run out of nucleotides.

  5. Take your DNA segments to the "sequencing chamber" at the front of the room. Place your sample in the proper "well" of the chamber. For example, the group which works with the reaction mixture containing dideoxyadenine will put its segments into "well A." Turn on the current (i.e., use your hands and mind!) and move your DNA segments their proper distance. Remember: Smaller segments migrate further than larger segments.

  6. When all groups have run their reaction mixture through the "sequencing gel," sketch the DNA sequence bands in the chamber illustration on the next page. Fill in the proper letters on the blanks to the right of the illustration. The sequence is read from bottom to top.

DNA Sequencing Gel

Discussion and Analysis Questions:
  1. To what part(s) of the reaction mixture are human hands analogous?


  2. The primer was just assumed to be in our "reaction mixture." What is the function of the primer?


  3. What role does the gel play in the sequencing process?


  4. Discuss several instances in which a person might want to make use of a DNA sequence.


  5. Why was some of the adenine in each reaction mixture radioactive?



Teacher Information

Background:
The Sanger sequencing method (named for Fred Sanger) involves enzymatic synthesis of radioactively labeled fragments of DNA from unlabeled DNA strands. The other DNA sequencing method (named for Allan Maxam and Walter Gilbert) involves the chemical cleavage of pre-labeled DNA segments in four different ways to form four collections of labeled fragments. Sanger, Maxam, and Gilbert received the Nobel Prize for their sequencing methods in 1980. The teacher should read over and provide the students with a reading on the Sanger sequencing method. Two sources are listed at the end of this activity.

Materials:

A. Pop-beads: Pop-beads can be ordered from Carolina Biological.

This lab is designed for four groups of six students. This requires 300 beads of each color. If smaller lab groups are desired, purchase three of each bag of beads so that you will have 600 of each color. You will have some beads left over.

Use wire cutters (or heavy scissors) to snip off the "stubs" from 25 of each color to create the dideoxynucleotides. Count out the pop-beads as listed in the student section. Use red fingernail polish to paint red "radioactivity" dots on half of the adenine beads.

Label four small baggies and put the proper beads into them. Put the four small baggies into a large baggie, and label the reaction mixture. You will need four large baggies and 16 small baggies.

Procedure:

  1. Fill in the chart as follows:

    3' G A C T G A A G C T G A 5'

  2. Assemble the beads in the same order as above. The "stubs" should point to the left.

  3. Run sequences through the pretend "gel."

Discussion and Analysis Answers:

  1. Human hands are analogous to the DNA polymerase at the beginning of the activity. They also represent the electric current as the fragments are separated.

  2. The primer initiates the replication of the templates by the DNA polymerase.

  3. The gel sets up an "obstacle course" which allows for segment separation. Smaller segments of DNA move further than the larger segments of DNA.

  4. DNA sequencing is used to "decipher" the sequence of genes which cause diseases (such as Huntington's disease), to sequence genes which code for necessary proteins (such as insulin), and in breeding studies which seek to breed genetically different, yet endangered, species.

  5. Radioactive adenine was added so that the film in the autoradiogram would be exposed and reveal the DNA sequence.

References:
Cooper, N. (editor). 1992. Los Alamos Science # 20: The Human Genome Project. 151-159. (Available from Los Alamos National Laboratory, Los Alamos, NM 87545)

Micklos, D. & Freyer, G. 1990. DNA Science. Cold Spring Harbor Press. 80-83. (Available from Carolina Biological, Burlington, NC 27216)


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