From DNA to Organism: A Study in DNA Function for the High School Biology Classroom
 
 

Modules 2: DNA and genes 

Alternate procedures for examining plants' DNA for the mutation of the AKT-1 gene

           The following is an alternative DNA isolation protocol. It is easier to use and quicker, but does not yield as clean a product from the extraction.  (Note to teachers: The students would not get both protocols, only the one that you, as instructor, have chosen for their class to use.)

     Part I. Getting out the goods

           Here, we use a commercially available kit to extract DNA from plants, the Cartagen DNA Isolation Kit .

           The first step in investigating the DNA of an organism is getting it out of the cells. Remember that the DNA of most organisms is kept in the nucleus of the cell so we need to open up both the cell and nuclear membranes to get at it. The biggest problem that we will face is the fact that the cell and the DNA inside of it is so small. We cannot just reach into the cell to get it. The other problem we will face is that there are a lot of chemicals in the cytoplasm that will breakdown the DNA if given the chance. Luckily scientists have figured out a way to get out just the DNA using a number of different chemicals. We will be using one such method today.

Follow the steps below. To help you keep track of where you are, check off the boxes as you go.

[ ]1.First we need to get some plant cells to get the DNA from. Each lab group has been provided with two plants to get their DNA from, one wild type (WT) and one mutant (MUT). For each of the plants we need to carefully obtain 50 mg (fresh weight) of leaves. To get them cut carefully at the base of the leaves and place them into a weighing dish. Make sure to label the two different samples.

WHY? The leaves are the easiest of the plant tissues to get DNA from. 
 

[ ]2.Use a clean razor blade to cut the leaves into smaller pieces, about the size of an eraser head from a pencil. Make sure to use a new razor blade for each of the samples. If you used the same razor, you could end up with contamination from your other sample.

WHY? The smaller the pieces, the easier it will be to grind it up.

[ ]3.Label the cap of one of the tubes either MUT or WT depending on whether you are working with the mutant plants or the wild-type plant samples. Place the grinding column into the collection tube and carefully place your plant samples into the grinding column.

WHY? The grinding column has a rough surface on the bottom that will help us to break up the cell walls of the plant cells to get at the DNA inside.  

[ ]4.Using a pipette, add 50ml of lysis buffer to the grinding column with the plant pieces in it.

WHY? The lysis buffer is specially designed to break apart the lipid bilayers that make up the cell and nuclear membranes, allowing for the DNA to be released.  

[ ]5.Slide the grinding tool into the grinding column and rotate the tool against the bottom of the column using moderate pressure. Do not push too hard or the column may break. Grind the plant sample for 30 seconds.

WHY? The grinding tool also has a rough surface that will help by rubbing against the rough surface of the grinding column helping to break up the cell walls.

 

[ ]6.After 30 seconds of grinding, add 150ml more lysis buffer to the column and put the grinding tool back into the column. Gently grind the sample for 15 seconds.

WHY? The lysis buffer helps to further break apart the membranes in the cell.

[ ]7.Remove the grinding tool and discard it (save the tube and grinding column!). Place your sample into the centrifuge and spin for 30 seconds.

WHY? The centrifuge will pull all of the liquid through the grinding column and also takes the solid debris from the cell (membranes, etc.) and pulls it all to the bottom of the tube separate from the DNA which is dissolved in the solution.

[ ]8.Remove your sample from the centrifuge and take out the grinding column. Discard the tool and column but keep your sample tube!

WHY? We already ground up the plant and no longer need the solid parts left in the tube or the tube itself. The DNA is dissolved in the solution in the collection tube.  

[ ]9.Close the cap on your tube and place it into the 95°C bath for 5 minutes.

WHY? The heat will help to let the cell lysis buffer break up the cells.

[ ]10.After the 5 minutes in the bath, remove your tube from the centrifuge and spin it again for 30 seconds.

WHY? Again, spinning will help to move the bigger materials down to the bottom of the tube so that we can get the liquid (which contains the DNA) separated from the waste.  

[ ]11.After the second spinning, remove your tube from the centrifuge and uncap it. Using a micropipette suck off the liquid on the top of your tube and put it into a new sample tube. Be careful not to suck up any of the solids at the bottom of the tube.

WHY: The leaves liquid is where the DNA is, the solids at the bottom are just the debris. This is a good way to separate the two.  

[ ]12.Using a marker, label the tube with your group’s initials and the sample ID, WT or MUT. Place these tubes into the freezer for storage until next class.

WHY? Freezing the DNA will preserve it until the next class when we will make copies of it.

 

     Part II. Making Copies of the Suspect

           Strangely enough all of the work that we did in the last portion of the lab didn’t produce enough DNA for us to be able to use it easily. This was a problem with DNA research for many years until in 1988 Dr. Kary Mullis, a surfer and scientist, came up with a revolutionary way to solve the problem. He created a process called PCR, or polymerase chain reaction, which is a way to make millions of copies of a segment of DNA in only a few hours. In this segment of the lab we will utilize Dr. Mullis’ discovery to make some copies of the gene in question. Along the way, you will be given some explanations of how the process works.

Follow the steps below. To help you keep track of where you are, check off the boxes as you go.

[ ]1.Obtain a pair of PCR reaction tubes from your instructor. For each of the tubes obtain 15ml of PCR cocktail from your instructor. The cocktail contains the following ingredients:

  • Water: This provides a liquid environment for the reaction to take place
  • PCR buffer: To help keep the reaction pH at the correct level
  • Taq DNA polymerase: This is the enzyme that assembles free DNA nucleotides in the solution into new strands of DNA. It comes from a hot-spring bacteria called Thermus aquaticus (thus the name Taq). This allows the enzyme to work at a wider temperature range, specifically at higher temperatures (remember that enzymes tend to be very specific about what conditions they will work in).
  • dNTPs: These are DNA nucleotides that are not bonded to a strand yet. They consist of a phosphate group, nitrogen base (A,T,C, or G) and a deoxyribose sugar. Nucleotides are the monomers (building blocks) of the DNA polymer. You are basically including a soup of adenine, thymine, cytosine and guanine nucleotides for the Taq to use to make new DNA strands.
  • MgCl2: This helps the Taq polymerase to assemble new DNA strands.
  • Primers: These are short, single-stranded segments of DNA that correspond to sequences on the DNA strand to be copied. One primer corresponds to one end of the segment, the other primer to the other end on the opposite strand. The primers that we use are specific to the mutant gene AKT-1, or the sequence of DNA inserted into the AKT-1 gene that disrupts the gene, causing the mutation. The insertion is referred to as a “ T-DNA .”  Use of primers specific for the T-DNA will yield a product if the DNA template has this mutation; i.e. the T-DNA insertion.

[ ]2.Take your sample of DNA from the extraction and add 10ml of it to the “cocktail” that we prepared in the previous steps. Your DNA will act as the template for the construction of the new DNA strands.

[ ]3.Label your tube so that you know which sample is the wild-type DNA (WT) and which is the potential mutant DNA (MUT). Also label your tube so that you know that it comes from your group. All of the groups will put their tubes in the PCR machine at the same time, so you will need to be able to find yours!

[ ]4.Place your PCR tube in the machine to have the reactions take place.

[ ]5.PCR works by raising and lowering the temperature in a series of steps and then repeating the process. The temperatures and times at each make up the PCR profile. Copy the profile for your PCR reaction below.

[ ]6.Your reaction will take a few hours to run so we will need to leave it overnight and check it the next morning.

     Part III. Who Has the Gene?

           Now that we have made millions of copies of the gene in question, we need to see whether or not the gene is in the mutant plant. The problem is that we have a soup of DNA in our tubes along with the copies that we made in the PCR reaction. Remember that Arabidopsis has ten chromosomes (5 pairs), with a total of about 125 million base pairs, but we only wanted to look at a small segment of one of the chromosomes, about 900 base pairs long. We need a way to separate out the small fragment from the longer strands of DNA in the mixture. Gel electrophoresis is just such a process.
           
           Gel electrophoresis uses electricity and a Jello-like substance called agarose to separate out molecules based on size. First a gelatin block is made out of agarose. The sample of molecules, DNA in our case, is placed in holes in one end of the block. An electric current is then applied to the block with the DNA in it. The DNA is pulled through the gel by the electricity. The smaller pieces of DNA can move more quickly through the gel than the bigger ones can and that is how we separate them!
           
Follow the steps below. To help you keep track of where you are, check off the boxes as you go.

     Making the Gel

[ ]1.Measure out 1.5 of agarose on a weighing dish using a scale.

[ ]2.Measure out 150ml of TAE buffer using a graduated cylinder. Pour the TAE into a 250ml flask.

[ ]3.Pour the agarose into the flask with the TAE. Gently swirl the flask to mix the agarose into the liquid.

[ ]4.The agarose will not go into solution just from swirling; we will need to heat it up to get that to happen. Place the flask into the microwave and heat on high for 1 minute, watching the flask as it heats. It is important to watch the flask because when the solution begins to boil, it will boil out of the flask and make a mess. If you see the flask begin to boil, stop the microwave and go to the next step.

[ ]5.After 1 minute, or when the solution begins to boil, carefully remove the flask from the microwave and gently swirl the solution in the flask. Look to see if you can still se particles floating in the solution. If so, continue to heat the flask 15 seconds at a time watching carefully so that it will not boil over. If the solution is totally clear and you cannot see any particles swirling in the mixture, remove the flask from the microwave and let it cool on the countertop for 3 minutes.

[ ]6.Once the liquid has cooled a bit, but not so much that it has solidified, you are ready to pour your gel into the mold. Place the plastic “combs” into one end of the gel. Make sure that they are pushed all the way into the gel mold so you get nice deep wells to put your samples into.

[ ]7.Set up your gel molds so that they will hold the liquid. Some molds need to be taped closed, others need only be placed into the running block sideways. Check with your instructor for how to get your molds ready to be filled.

[ ]8.Once the gel mold is ready to be filled you may begin filling your mold.   Pour the agarose/TAE solution into the mold carefully. You want to pour enough to just reach the top of the teeth of the comb, but not over them. The combs will make wells in the gel so you can put your samples in. Once the gel is poured wait 10 minutes to let the gel harden.

[ ]9.After 10 minutes, check to see that your gel is hardened completely. You can do this by touching it lightly with your finger. The gel should feel firm and have no liquid on or in it. If the gel is not hardened, wait a few more minutes and check it again.

[ ]10.If the gel is hardened, grasp the comb and slowly pull the comb out of the gel. It is very important that you not only pull slowly but also pull straight up out of the gel to keep your wells intact.

     Loading your Sample into the Gel

[ ]11. Place your gel into the gel electrophoresis chamber. The wells of your gel should go on the end of the chamber with the black (negative) wires. This is important because DNA is slightly negatively charged and will be attracted to the positive end of the chamber once the electricity is turned on.

[ ]12.Obtain a new tube for each of your samples and one extra tube for a new sample that you will run with your DNA samples. For each of your DNA samples place 30ml of it into a new tube, and label the tubes accordingly.   Return your PCR samples back into the freezer for future use.

[ ]13.Use a pipette to add to it 10ml of 100 base pair DNA ladder solution to the extra tube. This is a solution that has pieces of DNA of known size in it so that we can estimate the size of our DNA in the gel after we run it.

[ ]14.Before you load your samples you need to add a little buffer (with dye) to them so that you can see them. Using a pipette add 5ml of loading buffer to each of your samples including your DNA standard. Mix the solution by gently pipetting up and down.

[ ]15.You are now ready to load your samples into your gel. For each of the samples, take all of the liquid out of the tube and carefully pipette it into one of the wells of the gel. Try to put your samples as close to the center of the gel as possible to get the best results that you can. Record the position of your samples in the table below so that you will know which is which.

[ ]16.Once the samples are loaded, you can place the lid on the electrophoresis chamber and plug it into the power source. When you are ready, turn the power on. Ask your instructor for the voltage to set your power source at for your electrophoresis. Record the voltage and time for your electrophoresis run below. The reaction will take a while to happen.

Electrophoresis Run Data


  Voltage _________              Time _________

     Staining your Gel

           Another problem with DNA is that even in such large amounts it is not visible to the human eye. Scientists need to use dyes that will stick to the DNA to be able to see it. Many of the dyes that stick to DNA are harmful chemicals and need to be handled with great care. Luckily for us, we are going to use a slightly less dangerous chemical, methylene blue. As with the previous steps, check off the steps as you go.

[ ]1.Get a DNA staining tray and pour enough DNA stain into the tray to fill it approximately 2 cm deep.

[ ]2.Carefully remove your gels from the electrophoresis rig using a spatula.   Place your gel into the DNA staining tray. Make sure that the dye is deep enough to just cover your gel. Leave the gel in the dye for 30 minutes. The methylene blue will diffuse into the gel and stick to the DNA molecules that it runs into.

[ ]3.After 30 minutes, remove your gel from the stain and place it into the water bath. When you take it out the gel will probably be very dark blue, don’t worry if you can’t see any DNA yet. It will remain in the water to “de-stain” overnight. As the gel soaks in the water the methylene blue will diffuse back out of the gel and into the water. The dye molecules that have stuck to the DNA will not be able to diffuse out, leaving bunches of dye where there is DNA in the gel.

     Interpreting your Results

Now we have DNA bands to look at, but what does it mean?

[ ] 1.Remove your gel from the water tray and place it carefully on the light table. With the light table on, you should be able to see blue bands in your gel. Each of these bands corresponds to a segment of DNA.

[ ]2.In electrophoresis, the smaller the segment of DNA, the farther it will travel. So the farther from the well a band is, the smaller the piece of DNA it represents. Sketch your DNA bands into the gel diagram below.  Include in your diagram the DNA standard bands; they will help us in the next step.



[ ]3.Use the diagram provided by your teacher of the DNA standard to label the segments of the DNA standard in your diagram above. Start with the smallest segment and work your way towards the smaller segments.

[ ]4.You can use this information to estimate the size of the bands in your DNA samples, and to see if they have the AKT-1 gene.