WebCutter Lab

There are three modules in this activity.  In the first module you will learn about DNA sequencing.  this is a paper-and-scissors exercise rather than a lab activity because DNA sequencing in the lab either involves the use of radioactive materials or very expensive chromogenic materials.  Still, this DNA sequncing activity should help you understand the technique and science of DNA sequencing.  After you learn the basics of DNA sequencing, you will read a DNA sequence from a mock autoradiogram.  This DNA sequence is from a gene fragment that has been cloned into the plasmid pWEBCUTTER.

In the second activity, module 2, you will go on-line to analyze your mystery DNA sequence.  Use the internet address http://www.carolina.com/webcutter  to access the Carolina Webcutter softward.  The Webcutter software was originally designed for use by researchers.  However, Carolina Webcutter is a custom version that has been designed for your use.  There is introductory text and hypertext to guide you through the process of DNA analysis.  During the course of this analysis, you will use a link to the National Center for Biotechnology Information (NCBI) to access a DNA sequence database analysis program called BLAST.  The sophisticated BLAST software is the same software used by scientists today in their research.

Based on your work in Module 1 and Module 2, you will design and execute an experiment to determine whether the pWEBCUTTER DNA actually contains the gene predicted by BLAST.  This will be Module 3.

Module 1:  DNA sequencing

Determining the DNA sequence of a gene is a critical step in identifying that gene.  How is this done?  The most commonly used method today employs compound called chain terminators.  The word "terminator" describes the inhibitory effect these compounds have on DNA polymerase, the enzyme that synthesizes DNA.  Chain terminators allow us to look over the shoulder of DNA polymerase to see the order in which DNA polymerase adds bases to a new DNA strand.  With this information we can deduce the sequence of the template strand of DNA that DNA  polymerase is using as a pattern to synthesize the new strand of DNA.

So what are chain terminators, and how do they block DNA synthesis?  Chain terminators are very similar to normal nucleotides, but they lack an important feature of nucleotides:  the 3' hydroxyl group.  This 3' hydroxyl group is required to form the bond with the next nucleotide in the DNA strand.  Without it, DNA synthesis cannot proceed.  When DNA polymerase adds a chain terminator to a DNA strand, rather than a normal nucleotide, DNA synthesis is terminated at that point.

In a sequencing reaction, the DNA you want to sequence is mixed with primers (the small pieces of DNA needed to jump-start DNA polymerase) DNA polymerase and the four different nucleotides (A, T, C, G) one of which is radioactive (but otherwise normal).  This reaction is then subdivided into four smaller reactions, and each oof the four small reaction is spiked with different chain terminator:  one that can terminate the reaction at A's (adenine), one that can terminate the reaction at T's (thymine), one that can terminate the reaction at C's (cytosine), or one that can terminate the reaction at G's (guanine).  The DNA polymerase present in these four reactions, synthesizes new DNA strands from the original DNA molecule.  Occasionally, however, a chain terminator is inserted in the place of the normal nucleotide, and the synthesis is terminated on either A, T, C, or G, depending on which chain terminator is present in that reaction.

After the synthesis reactions are complete, the A, T, C, and G reactions are separated by size on an acrylamide gel that is capable or resolving very small fragments of DNA.  After gel electrophoresis, the acrylamide gel is dried and exposed to film.  Because one nucleotide in the reaction was radioactive (although otherwise normal), the DNA fragments show up as bands on the film.  This film is referred to as an autoradiogram.  The sequence of the original DNA is read from the autoradiogram by starting at the bottom of the film (with the shortest DNA fragment) and reading upward.

There are newer and more recent variations on how sequencing is done.  Sequencing can be performed using a thermocycler and Taq polymerase instead of DNA polymerase.  In addition, instead of being performed with one radioactively labeled nucleotide, sequencing can be performed using chain terminating nucleotide labeled with dyes.  Because each different terminating nucleotide (A, T, C, G) is labeled with a different color dye, all of the terminating nucleotides can be placed in the same reaction and run in one lane on an acrylamide gel.  When this method of sequencing is used, the DNA fragments are run off the gel.  As the fragments run off the gel, a laser connected to computer software is used to identify which dye that particular DNA fragment is labeled with.  This allows the computer to determine which terminating nucleotide (A, T, C or G) that particular DNA fragment end with.  In this way an entire DNA sequence is complied.  The equipment to do this type of sequencing is very expensive, but the method is efficient and is especially useful is one is doing large amounts of sequencing.  For an animated description of this method of sequencing, see http://vector.cshl.org --> resources --> biology animation library --> cycle sequencing.

Part 1

1. Divide into four groups, with each group at a workstation.
2. Your group will be either the adenine, thymine, guanine, or cytosine group.
3. Cut out the DNA sequence strips from the DNA sequence page.  There should be six DNA sequence strips on your page.
4. Place you DNA sequence strips into a plastic tray.  These trays represent the test tubes with the sequencing reactions.  Each test tub will yield a result for one of the nitrogen-containing bases in DNA (A, T, C, G).
5. Cut your DNA sequence strips at "your" base.  Each DNA sequence strip should have only one cut in it.  Place the end with the black box (representing the primer) back in the weigh boat.  The other piece should be discarded.  When you are finished, you should have six pieces of DNA sequence strips (with the black box) of all different lengths.   Note:  No cutting of DNA strand occurs in an actual sequencing reaction; instead there is a termination of synthesis caused by a lack of the 3' hydroxyl group on the chain terminator.
6. Arrange your fragments on the sequencing gel chart.
7. Read and record the DNA sequence of the gel.

Part 2

1. You should have a mock autoradiogram, representing the results obtained when a radioactive sequencing gel is exposed to film.
2. Put your mock autoradiograms against a white background (such as a piece of plain white paper) so you can easily see the bands.
3. Read and record the DNA sequence from your autoradiogram, starting from the bottom up.  It will be harder to read the top portion of the gel, because the bands get closer and closer together.  On a real sequencing gel, it is possible to read only so far from the primer before the bands become too close together.

Module 2:  Bioinformatics

Part 1

Go to the Internet address http://www.carolina.com/webcutter

Read the background information on "How do I use Webcutter?"

Since you have a DNA sequence that you would like to know the identify of, you should choose the link on this webpage to go to the NCBI (National Center for Biotechnology Information) homepage.  This link is located under the heading "If you have a DNA sequence and want to know its identity."  At the NCBI homepage, select the BLAST button on the toolbar at the top of the page.  This will take you to the BLAST page, where you should select "Standard-nucleotide-nucleotide BLAST (blastn)" under the Nucleotide BLAST heading.

This will take you to the Basic BLAST page where you will input your sequence.  Leave all the settings on their default settings (i.e., don't change any settings) and enter your sequence into the box.  Type in approximately 50 nucleotides from your mystery sequence.  It is not necessary to type in the entire sequence; however, the more nucleotides that are typed in, the more accurate the match BLAST will obtain.  Then, click the BLAST button.  To see your results click the FORMAT button.

The BLAST program searches for similarities between the sequence submitted and all the other sequences in its database.  It will show multiple sequences that have homology with the original sequence.  It will score these matches on the basis of sequence similarity.  The Color Key for Alignment Scores and the e values refer to the amount of homology between the original sequence and the matches.  The first entry under "sequences producing significant alignments" is the sequence with the greatest homology to the entered sequence.  Click on the entry to see the full record. 

For more information on BLAST, click on the BLAST FAQ's and other information links on the "results of BLAST" page.

Part 2

Design an experiment to verify whether or not the BLAST prediction is accurate.

Your resources include a tube of pWEBCUTTER plasmid DNA, EcoRI, PstI, and the Carolina Webcutter on-line software.

Predict the sizes of the restriction fragments that you expect to see after restriction digestion of pWEBCUTTER with EcoRI and PstI.  Create a table to keep track of the fragment sizes.

Module 3:  Restriction Analysis and Gel Electrophoresis

Procedure A:  Set Up Restriction Digests

1. Label three 1.5mL tubes for the restriction reactions as follows:  E for EcoRI, P for PstI, and U for uncut.
2. Use the table below as a checklist while adding reagents to each reaction.  Use a fresh micropipet tip for each reagent.  All groups will share the same EcoRI and PstI at a central station.
3. Mix the DNA and enzymes by pipetting up and down several times.  Use a fresh tip when adding DNA into each reaction tube to prevent cross contamination of enzymes.
4. Incubate all the reaction tubes for a minimum of 20 minutes at 37ºC. 

Note:  The total volume of each reaction is 10μL.

Procedure B:  Cast Agarose Gel

1. Seal the ends of a gel-casting tray with masking tape or rubber ends and insert a well-forming comb.  Place the gel-casting tray out of the way on the lab bench, so that the agarose poured in the next step can set undisturbed.
2. Carefully pour enough agarose solution into the casting tray to fill it to a depth of about 5mm.  The gel should cover only about 1/3 the height of the comb teetch.  While the agarose is still liquid, a pipet tip or toothpick can be used to move large bubbles or solid debris to the sides or the end of the tray.
3. The gel will become cloudy as it solidifies (about 10 minutes).  Do not move or jostle the casting tray while the agarose is solidifying.
4. When the agarose has set, remove the tape or rubber ends from the ends of the casting tray and place it on the platform of the gel box so that the comb is at the negative (black) end.
5. Fill the gel chamber with tris-borate-EDTA (TBE) buffer to a level that just covers the entire surface entire surface of the gel.
6. Remove the comb by gently pulling up.  Be careful not to tear the wells.
7. Make certain that the sample wells formed by the comb are completely submerged.  If you notice dimples around the wells, slowly add buffer until the dimples disappear.
8. The gel is now ready to load with DNA.

Procedure C:  Load Gel

1. Add 2μL loading dye to each reaction tube.  Mix the dye with digested DNA and pool the liquid in the bottom of the tube by tapping the tube on a lab bench, or with a 10-second spin in a microcentrifuge.
2. Use a micropipet to load the contents of each reaction tube into a separate well in the gel.  Use a fresh tip for each reaction tube.
3. Load 10μL of the DNA ladder into one lane of each gel.

Gel loading tips

• Use two hands to steady the pipet over the well.
• Be careful to expel any air in the micropipet tip end before loading the gel.  (If an air bubble forms a "cap" over a well, DNA/loading dy will flow into the buffer around the edges of the well.
• Dip the pipet tip through the surface of the buffer, position it over the well, and slowly expel the mixture.  The sucrose in the loading dye makes the sample more dense than TBE buffer and causes the sample to sink to the bottom of the well.  Be careful not to punch the tip of the pipet through the bottom of the gel.

Procedure D:  Electrophorese

1. Close the top of the electrophoresis chamber and connect the electrical leads to an approved power supply, anode to anode (red-red) and cathode to cathode (black-black).  Make sure both electrodes are connected to the same channel of the power supply.
2. Turn the power supply on and set the voltage to 85V.  Shortly after the current is applied, you should see loading dye moving through the gel toward the positive pole of the electrophoresis apparatus.
3. Allow the DNA to electrophorese until the bromophenol blue band from the loading dye nears the end of the gel.
4. Turn off the power supply, disconnect the leads from the inputs, and remove the top of the electrophoresis chamber.
5. Carefully remove the casting tray and slide the gel into the staining tray labeled with your group name.  Stain your gel.

Data Analysis:  Does pWebcutter contain the DNA fragment found on your autoradiogram?

1. Place your stained gel on a white-light box.  Orient it so that the wells are at the top of the gel.
2. The DNA ladder has fragments of 1857bp, 1058bp, 929bp, 383bp, and 121bp.  The 121bp fragment will probably not be visible.  Measure the distance, in mm, that each marker fragment migrated from the sample well.  Measure from the front edge of the well to the leading edge of each band.
3. Set up semi-log graph paper with distance migrated at the x- (arithmetic) axis and base-pair length as the y- (logarithmic) axis.  Plot the distance migrated versus the base-pair length for each marker fragement.  Connect the data points with a line.  Because you are plotting base-pair length (as the equivalent to molecular weight) on the logarithmic axis, it is not necessary to take the log of the fragment sizes before graphing.
4. Measure and record the distances migrated by the various restriction digest fragments.  Next, to determine the sizes of each fragment, first locate the distance it migrated on the x-axis.  Then, use a ruler to draw a vertical line from this point to its intersection with the marker data line.  Now, extend a horizontal line from this point to the y-axis.  The number on the y-axis is the calculated base-pair size of the fragment.
5. Compare the sizes you obtained to the sizes you predicted on the basis of the Webcutter analysis of the restriction enzyme map.  Do they match?  Did your plasmid contain a fragment of your autoradiogram DNA?

Questions

1. How much plasmid DNA was in the EcoRI digest?  How much plasmid DNA was in the PstI digest?  The pWEBCUTTER DNA is supplied at a concentration of 0.5μg/μL.
2. Why is it necessary to have more DNA in the PstI digest?
3. How much DNA is present in the different DNA bands?  There are 4240bp total in the pWEBCUTTER plasmid.  Create a table to display your results.
4. Where the 120bp band from the PstI digest the and 121bp band from the DNA ladder visible on the gel?  Why or why not?  What about the 163bp band?