Lab 11: Restriction Enzyme Cleavage of DNA and Electrophoresis

Overview:
Molecular biologists use many techniques to analyze DNA. In this lab, you will utilize several of these techniques to study the DNA of a lambda bacteriophage. Restriction enzyme digests of phage lambda DNA will be used to demonstrate techniques for separating and identifying DNA fragments using gel electrophoresis.

Objectives
1. Demonstrate how restriction enzymes are used in genetic engineering. 2. Use electrophoresis to separate DNA fragments 3. Determine unknown DNA fragment sizes when given fragments of known size.

Introduction
Restriction endonucleases are essential tools in recombinant DNA methodology. Several hundred endonucleases have been isolated from a variety of prokaryotic organisms. In nature, an endonuclease is a prokaryotic cells defense against foreign DNA inserted by a virus. The endonuclease cuts DNA at palindromes sites. Restriction endonucleases such as EcoRI and HaeII are named according to a specific system of nomenclature. The letters refer to the organism from which the enzyme was first isolated. The first letter of the name stands for the genus name of the organism. The next two letters represent the second word of the species name. The fourth letter (if there is one) represents the strain of the organism. Roman numerals indicate whether or not the particular enzyme was the first isolated, second, or so on. Examples: EcoRI E co R I = genus Escherichia = species epithet coli = strain RY13 = first endonuclease isolated HaeII H = genus Haemophilus ae = species aegyptius II = second endonuclease isolated from organism

These restriction endonucleases recognize specific DNA sequences in double stranded DNA (usually a 4-6 base pair sequence of nucleotides) and digest the DNA at these sites. The result is the production of fragments of DNA of various lengths corresponding to the distance between identical DNA sequences within the chromosome. Some restriction enzymes cut cleanly through the DNA helix at the same position on both strands to produce fragments with blunt ends. Other endonucleases cleave each strand off-center at specific nucleotides to produce fragments with over hangs or sticky ends. By using the same restriction enzyme to cut DNA from two different organisms, complementary sticky ends will be produced and can realign in a template-compliment manner allowing DNA from two sources to be recombined. By taking DNA fragments and systematically reinserting the fragments into an organism with minimal genetic material, it is possible to determine the function of particular gene sequences. In this way, the genome or chromosomal character of the organism can be dissected, rearranged, and tested for function and organization. Further, when a gene sequence of interest has been identified, it is possible to remove it from a chromosome and insert it into a plasmid that is capable of generating many copies of itself and the foreign gene sequence. This process is known as gene cloning. Finally, the sequence can be transferred to other systems to produce a desired effect as done during the pGLO transformation lab. Another well-known example of genetic engineering is the cloning of the human insulin gene. The gene was removed from human chromosome number 11 using restriction endonucleases and was inserted into an E. coli plasmid at a site that was cut with the same restriction enzyme as was used to cut out (excise) the human insulin gene. Restriction endonucleases and gene cloning have made possible the large-scale production of human insulin in the laboratory. 1

Electrophoresis is a technique that is used to separate different sized fragments of DNA. When a molecule enters an electrical field, the mobility (speed) at which the molecule will move is influenced by the charge of the molecule, the strength of the electrical field, the size and shape of the molecule, and the density of the medium (gel) through which the molecule is moving. Consequently, it is possible to separate heterogeneous groups of molecules. When all molecules are positioned at a uniform starting point on the gel and the gel is placed in a chamber containing a buffer solution and electrodes, the molecules will migrate and from bands (concentrations of homologous molecules). Nucleic acids, like DNA and RNA, move because of the charged phosphate groups in the backbone of the DNA structure. Because the phosphates are negatively charged at neutral pH, the DNA will migrate through the gel toward the positive electrode. In this lab, we will use an agarose gel. The density of the agarose gel can be varied to improve the resolution of similarly sized molecules. In agarose, the migration rate of linear fragments of DNA is inversely proportional to their size. The smaller the DNA molecule, the faster the piece migrates through the gel and the farther it will move toward the positive electrode in a specific amount of time. The size of the fragments produced by a specific endonuclease (EcoRI will be used in this lab) can be determined by using standard fragments of known size. The fragments of the standard, control sample (also called the ladder) were created using the restriction endonuclease Hind III. The control and the unknown samples of DNA cut with Eco RI and Pst I will be run simultaneously on the electrophoresis gel. Staining DNA with a dye called methylene blue or ethidium bromide will allow visualization of bands of DNA. Ethidium bromide is a fluorescent pink dye that must be viewed under UV light and it is also a carcinogen, so we will not use ethidium bromide in class! A standard curve (for Hind III digest) can be plotted using the distance each band migrated (x-axis) measured in millimeters and the known size of the standard fragments on the y-axis. The size of the fragments is expressed as the log of the number of base pairs they contain; this allows the data to be plotted as a straight line (see Figure 11.1). The migration distance of the unknown, plotted on the x-axis, will allow fragment size to be determined based on the standard curve (control). Therefore, the size of the fragment (y-axis) scale will be logarithmic and special semi-log graph paper will be used to construct the graph for this experiment. Figure 11.1: Effect of distance fragment migrated on size of DNA fragment using a specific restriction enzyme
Many Unknown fragment #1 Known fragment size of standard Hind III digest Unknown fragment size

Size of Standard Fragments (# base prs) Logarithmic Scale

Unknown fragment #2

Few Short Long

Distance Standard Fragments Traveled (mm)

DNA Fragment Separation Procedure I. Sample Preparation (work in groups of 4)

1. Label four microtubes L, P, E, and H and place them into the styrofoam microtube rack. L = No restriction enzyme - uncut Lambda DNA (Lambda DNA is the bacteriophage) P = Pst I restriction digest of Lambda DNA (from Providencia stuartii bacteria) E = Eco RI restriction digest of Lambda DNA (from Escherichia coli bacteria) H = Hind III restriction digest of Lambda DNA (standard*) (from Haemophilus influenzae) (* The fragment sizes are known for Hind III, thus the Lambda/Hind III is a size standard/ ladder)
1

2. Dial the micropipette to 10uL, add a clean tip, and transfer 10uL of Lambda DNA cut with Pst I enzyme from the P tube stock on ice to the P tube in your microtube rack. Be sure to replace the P 0 tube stock to the ice. 0 3. Repeat step 2 for the L, E, and H DNA tubes that are on ice. Be sure to use 10uL each time. Use a new tip for each solution. Place the stock tube back on ice immediately after withdrawing 10uL. Is the DNA added to each tube visible? In order to see the progress of the DNA separation on the agarose gel, we must add some visible dye to mark or track the movement of these invisible DNA fragments through the gel. This is accomplished by adding a blue dye called a loading dye. The loading dye actually contains two dyes: one dye that moves through the gel faster than the DNA fragments and another dye that moves slower than all the DNA fragments.

2 4. Redial the micropipette to 2.0uL and transfer this amount of loading dye to each of the four tubes marked L, P, E, and H in the microtube rack. Use a new pipette tip for each transfer. 0 5. The DNA and loading dye must be thoroughly mixed in each tube before placing the samples in the gel wells for electrophoresis. Using a microcentrifuge, place the four tubes from your microtube foam rack into the microcentrifuge, being sure to space them evenly. Have your teacher check before spinning the tubes. Pulse-spin the tubes (hold the button for a few seconds). This allows the DNA and the loading dye to mix.

II. Loading the Gel and Setting up the Gel Chamber for Electrophoresis
1. Add buffer solution to the electrophoresis chamber so that the solution covers the gel. Make sure the black wedges are removed from either side of the gel bed and take out the comb that was used to create wells as the agarose gel cooled and solidified. 2. Pipette 10uL from each tube (L, P, E, and H) into separate wells in the gel. Use a fresh tip for each tube. Load the gel in the following order: Well Orientation back / top front / bottom Lane # 1 2 3 4 Tube L P E H 3

MAKE SURE WELLS ARE LOCATED AT THE NEGATIVE ELECTRODE SIDE (BLACK) OF THE ELECTROPHORESIS CHAMBER!!

3. Close the lid of the electrophoresis chamber and connect the electrical leads to the power supply and to the electrophoresis chamber: anode to anode (red to red) and cathode to cathode (black to black). Make sure both electrical leads are attached to the same channel of the power supply. Have your teacher check your work BEFORE you turn on the power supply. 4. Electrophorese at 150 volts for 25 minutes. Shortly after current is applied, the loading dye can be seen moving through the gel toward the positive side of the gel chamber. 5. When electrophoresis is complete, turn off the power supply, disconnect the leads from the power supply and chamber, and open the lid of the electrophoresis chamber. Caution: buffer and gel may be HOT! 6. Remove the gel from the bed. The gel is very slippery and fragile. Hold the gel level and slide the gel off the bed into a large plastic mass boat. 7. Pour excess buffer back into its original container. Its recyclable and will be reused for subsequent electrophoresis experiments.

III. Staining Gel

What color was the DNA before you added the loading dye? Since DNA is not naturally colored, it is not immediately visible in agarose gel. Following electrophoresis, the DNA in the gel must be placed in a staining solution in order to visualize the bands of DNA. Remember that scientists look at DNA in a gel to determine whether the gene they are looking for was successfully cut out of the chromosome. One of the ways they identify the gene of interest is by the size of the gene. 1. Label the mass boat that now contains your gel with names of all group members and period number. 2. Wearing latex gloves, add enough DNA stain to just cover the gel. 3. Allow gel to stain overnight. Cover mass boat with gel and stain solution with plastic wrap. Properly dispose of the latex gloves. *end of day one* ----------------------------------------------------------------------------------------------------------------------------------

IV. Destaining Gel (DAY TWO)

The agarose gel absorbed the DNA staining solution used yesterday. At this stage, you will remove the excess DNA stain from the gel. Some stain will remain in the gel itself, but the DNA in the gel will be stained a darker blue color. After destaining the gel, your task will be to analyze the DNA banding patterns in the gel produced by digesting lambda DNA with different restriction enzymes. 1. Pour the DNA staining solution in the mass boat back into a beaker. Rinse your gel several times with tap water to destain the gel. 2. Fill your staining tray with enough water to cover the gel. Let stand for 10-20 minutes, and then pour off excess water. Examine the DNA band patterns in each lane. 4

V. Data Analysis
One of the first ways of analyzing your gel is to determine the approximate sizes of each of your restriction fragments. To determine the size of the fragments in each digest, compare the DNA restriction fragments to the DNA fragments of known sizes using the Hind III standard (H well). The standard is also called a ladder because the distance between the bands (rungs of the ladder) is uniform and known. See page 3 to recall the orientation and contents of each well on your gel. 1. Measure the distance (in millimeters, to tenths place) that each of the DNA fragments traveled from the well with the Hind III standard digest. Measure the distance from the bottom of the loading well to the center of each DNA band and record data in a data table created during pre lab preparation.

2. Create a graph as explained in the introduction on page 2 for the Hind III standard ladder. The size of the fragments (# base pairs) from largest to smallest fragment is the following sizes: 23,130bp; 9,416bp; 6,557bp; 4,361bp; 2,322bp; 2,027bp. (HINT #1: How do you know which fragment is the largest by looking at your gel?) (HINT #2: What is total size of Lambda DNA before being cut by Hind III enzyme?) The semi-log graph paper has three cycles or sections ranging from 1-10. The axis with the semi-log scale is the y-axis and should be labeled Fragment Size (# base pairs). Each cycle of 1-10 should start with a value 10 times greater than the previous cycle. Based on the information provided, we know that the number of base pairs of the standard fragments ranges from 2,000 to 48,000 so an appropriate scale might be: First cycle: 100 - 1,000 base pairs Second cycle: 1,000 - 10,000 base pairs Third cycle: 10,000 - 100,000 base pairs Try to connect as many points generated by plotting the distance traveled compared to the size of fragments for the Hind III standard ladder. Create a line of best fit for the standard data. 3. Now measure the distance of each band in the other three lanes L, P, E. Use the standard line created on semi-log paper from the Hind III digest to estimate the size of each band in base pairs. Be sure to include measurement distance as well as size of band in number of nucleotides in your data table.

VI. Conclusion: Points to Ponder

1. How were the size of fragments created from the Eco RI and Pst I digests determined? 2. Why was only one band visible from the well that contained the L solution? 3. The size of the fragments can be determined by creating a graph or by estimating length of unknown bands to those of standard ladder. Which technique is more accurate? Explain. 4. Which tube (L, E, P, H) is the positive control? Negative control? Explain your answers. 5. How many restriction sites exist in the Lambda DNA for the endonuclease Hind III. Explain.

VII. Lab Analysis Questions

1. Discuss how each of the following factors would affect the results of electrophoresis: a. Voltage used b. Running time c. Amount of DNA used d. Reverse polarity 2. Two small restriction fragments of nearly the same base pair size appear as a single band, even when the sample was run to the very end of the gel. What could be done to resolve the fragments? Why would it work? 3. What are restriction enzymes and how do they work? 4. What is the source of restriction enzymes? What is the function of restriction enzymes in their native organism? 5. Describe the functions of electricity and the agarose gel in electrophoresis. 6. What are the functions of the loading dye in electrophoresis? How can DNA be prepared for visualization? 7. If a restriction enzyme digest resulted in DNA fragments of the following sizes: 4,000 base pairs; 2,500bp; 2,000bp; and 400bp sketch the resulting separation by electrophoresis. Show the staring point, location of the positive and negative electrodes, and the DNA bands in the gel. 8. Use the graph you constructed for your lab to predict how far in millimeters a fragment of 8,000 base pairs would migrate from the well. 9. How can a mutation that alters an endonuclease recognition site be detected using gel electrophoresis?