eculab MolecuLab 45 DNA Sequencing - A Classroom Exercise Student Manual 950 Walnut Ridge Drive Hartland, WI USA

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1 Mo eculab DNA Sequencing - A Classroom Exercise Student Manual 950 Walnut Ridge Drive Hartland, WI USA Revision 11/2011

2 Student Manual BACKGROUND DNA sequencing technology was developed in the mid 1970s. From three methods were developed for the sequencing of DNA [1][2][3]. The first two sequencing technologies (named the plus-minus and wandering spot methods, respectively) were rather inefficient and involved the use of toxic chemicals. Because of these problems, they fell out of favor when the chain termination method for DNA sequencing was developed by Fredrick Sanger and Alan Coulson in 1977[3]. The chain termination method uses DNA polymerase and inhibitors that terminate synthesized chains at specific altered base residues. This chain termination method (or shotgun method) is the method currently used for sequencing of DNA and is described here. DNA sequencing is a process used to determine the exact order of nucleotides in a strand of DNA. This process requires the following ingredients: Single-stranded DNA template DNA primer DNA polymerase Modified nucleotides (dntps = datp, dttp, dgtp and dctp) Fluorescently labeled nucleotides (ddntps = ddatp, ddttp, ddgtp and ddctp) A primer sequence of DNA (~20-30 bases) is chosen to match a region upstream of the DNA template that is to be sequenced. Template region to be sequenced 3 5' 3 Upstream primer Polymerase elongates this strand by adding dntps or ddntps. 5 2

3 Student Manual In the sequencing reaction, the primers hybridize to their complementary region on many copies of DNA template. Then DNA polymerase elongates the primer by adding dntps to the 3 end. By chance, DNA polymerase may add a modified nucleotide (ddntp) instead of a normal nucleotide (dntp). When a modified nucleotide (ddntp) is added, the elongation process stops on that strand. The proportions of the ingredients are such that a modified nucleotide instead of a normal nucleotide will be added at each possible location across from the template. This creates DNA fragments that differ in size by a single nucleotide. These fragments can then be sorted using electrophoresis in a gel that can separate single base size differences. Modern methods can sequence DNA fragments approximately nucleotides long. Large Scale Sequencing methods such as the human genome project involve taking an entire genome and chopping it into smaller pieces. These pieces are sequenced and then overlapping fragments are assembled to determine the entire genome sequence. A DNA sequencer automates the DNA sequencing process. DNA sequencers have become more important due to large genomics projects and the need to increase productivity. Modern automated DNA sequencing instruments, or DNA sequencers, are able to sequence as many as 384 fluoresecently labeled samples in a batch (run) and perform as many as 24 runs a day. These perform size separations of individual bases and assemble the readings into a chromatogram. The magnitude of the fluorescent signal is related to the number of strands of DNA that are in the reaction. If the initial amount of DNA is too small, the signals will be weak. The chromatogram generated by the sequencer allows the researcher to determine the base sequence based on fragment size. The chromatogram is analogous to an agarose gel with a size marker, but with many more fragments being evaluated at once. Refer to the sample chromatogram below. Each peak is caused by the fluorescent signal from one of the four ddntps. 3

4 Student Manual Timeline 1975 The first complete DNA genome to be sequenced is that of a virus 1977 A method of DNA sequencing is developed and is the basis of modern methods 1987 First Automated DNA sequencing machine is commercially available 1990 Initiation of the Human Genome Project - a federally funded $3 billion initiative to determine the sequence of the human genome 2001 Draft of human genome sequenced 2008 Approximately 200 organisms sequenced (mostly bacteria, but including cow, pig, sheep, goat, chicken, rat, dog, cat, fruit fly, zebra fish, corn, wheat, rice, barley.) STUDENT INSTRUCTIONS Purpose Upon completion of this experiment, you should have gained a better understanding of the basic procedure for DNA sequencing. Pay close attention to the steps you are simulating in this sequencing reaction. This simulation should allow you to grasp the concepts of DNA structure, base complementarity. Materials per student: 1 strand of template DNA 12 white dntp (3 each A,T,C,G) 1 flourescent ddntp 1 3-base primer sequences Transparent tape Sequencing was a major accomplishment for the advancement of research. It is the basis for the field of biology called Genomics, which allows researchers to ask questions about how all of the organism s genes interact. Sequencing is used for researching specific genes that may be involved in cancer, addiction, diabetes, obesity and many other conditions. Without this major development it would be nearly impossible to decide which genes could be responsible for certain ailments. Sequencing brings science one step closer to being able to treat diseases in a customized manner. 4

5 Student Manual 1. Work in one of eight groups. 2. Each group should obtain four copies of DNA that will serve as an unknown template for a sequencing reaction. This piece of DNA has been copied many times by a technique called PCR (polymerase chain reaction). The complementary strand is not included here but would be present in the tube of an actual reaction. (The DNA would be denatured so it is single stranded.) In reality, the DNA would be much longer. 3. Each member of the group should place a copy (or two) of DNA template on the table and place a short DNA primer (3 bases long) so that it matches its complementary bases and is aligned anti-parallel to the DNA template. In reality, this primer would be ~20 bases long. 4. Obtain a set of deoxyribonucleotides (dntps = datp, dctp, dgtp, or dtcp). Also obtain several copies of one kind of fluorescentlylabeled di-deoxyribonucleotide (ddntps). Other groups in the class will receive the three other kinds of labeled ddntps. 5. Your group will simulate one of four reactions. You will be the enzyme DNA polymerase in the DNA sequencing reaction. Starting at the 3 end of the primer, you may place either a dntp or a fluorescentlylabeled ddntp (colored) across from the template DNA strand. If you add a fluorescently-labeled ddntp, the reaction stops and no more bases may be added to that strand. Note that you are building the new strand in the 5 to 3 direction. 6. Use tape to keep your newly built DNA strand together. This strand would naturally form hydrogen bonds to its complementary template DNA strand but you don t need to tape it to its complementary template because it will be denatured in step Repeat steps 5 and 6 until you have added your group s particular labeled ddntp at every possible location. 8. Denature your double stranded DNA into single strands. 9. When all groups are ready, load the contents of your group s reaction onto the gel. (This gel is made of polyacrylamide and is able to separate DNA strands that differ in size by a single nucleotide). 10. Electrophorese by moving each DNA fragment through the gel as far as the marker indicates. The marker represents the number of base pairs in each fragment. Note that shorter fragments migrate farther than longer fragments. 11. When all groups have migrated their DNA fragments, note which fluorescently-labeled dntp is at each position. 5

6 Student Manual QUESTIONS 1. A. Write the sequence of the newly synthesized strands as you read the gel from bottom to top, including 5 and 3 polarity. 5. What 3-base primer sequence could you use to sequence the strand that is complementary to the fragment on the gel you just sequenced? (Sometimes this is done to verify a double stranded sequence). B. Is the sequence pattern on the gel the same as the original template DNA sequence? 2. Which steps of an actual sequencing reaction does this activity simulate? 6. If the following strand serves as a template for a sequencing reaction, draw what the gel would look like. 3 TTCGACGGGCCAAAT5 M 13 A 12 T 3. Which steps of an actual sequencing reaction are not simulated in this activity? 11 G 10 G 9 C 8 C 7 C 4. What would be the sequence of a 3-base primer that you could use to sequence a fragment of DNA downstream of the sequence you just determined? 6 G 5 T 4 C 3 G 2 A 1 A 6

- In 1976 1977, Allan Maxam and walter Gilbert devised the first method for sequencing DNA fragments containing up to ~ 500 nucleotides.

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