Molecular Scissors: Lambda Digest Student Materials Introduction 2 Pre-Lab Questions. 5 Lab Protocol 6 Data Collection Worksheet. 9 Post-Lab Questions and Analysis.. 10 Plasmid Maps. 13 Last updated: August 7, 2017
Molecular Scissors Introduction Do you know what restriction enzymes are? Also known as restriction endonucleases, restriction enzymes are important enzymes that cut DNA at specific sequences called restriction sites. Restriction enzymes were discovered when scientists noticed that bacterial viruses (bacteriophages) can infect some bacterial cells and not others. The bacterial host cells that are resistant to infection produce enzymes that cut the invading viral DNA. The small fragments of viral DNA cannot be used to make new complete viruses stopping infection of the bacteria cell. Scientists named these enzymes restriction endonucleases or restriction enzymes for their ability to restrict or limit viral infection (see Figure 1). Figure 1. Bacteria defending themsleves against bacteriophages http://www.biomedheads.com/uploads/2/6/0/4/26041745/8703912_orig.jpg Since the discovery of the first restriction enzyme, hundreds of restriction enzymes that recognize and cut DNA at different restriction sites have been identified. The name of each restriction enzyme comes from the genus, species and strain of the bacteria that naturally produced it. For example, the enzyme EcoRI comes from: E = Eschericia genus co = coli species R = strain RY13 I = roman numeral I for the first enzyme indentified in the bacterium Most of the restriction ezymes used by scientists recognize restriction sites that are four to eight base pairs long. Let s look at a restriction site for EcoRI: 5 GAATTC 3 3 CTTAAG 5 Look closely at the sequences. Do you notice anything special? Read the top strand left to right outloud.now read the bottom strand right to left. Did you notice that you were reading the same sequence? This is called a palindrome and it is often a characteristic of restriction sites. In this case, EcoRI cuts between the G and the A when reading from 5 2
to 3. Because the enzyme cuts each of the two strands of the restriction site, it leaves staggered cuts that produce sticky ends. These short, unpaired sequences are the same for any DNA that is cut by EcoRI. This is sometime indicated in this manner: 5 G/AATTC 3 5 G AATTC 3 or 3 CTTAA/G 5 3 CTTAA G 5 While not as common, some enzymes like HaeIII cut both strands of DNA at the same position, producing blunt ends. 5 ATCAGG/CCATT 3 5 ATCAGG CCATT 3 3 TAGTCC/GGTAA 5 3 TAGTCC GGTAA 5 The ability of restriction enzymes to cut DNA at known sites make them an important tool in biotechnology. Scientists can cut open a DNA sequence and insert another sequence into the opening (Figure 2). Figure 2. Using restriction enzymes to create recombinant DNA Original double-stranded DNA molecule with a single restriction site for EcoRI 5 3 3 GAATTC CTTAAG 5 restriction enzyme site Cut with restriction enzyme Fragments of original DNA molecule produced by digestion with EcoRI and double-stranded DNA to be inserted into original DNA molecule. 5 3 G AATTC CTTAA G 3 5 5 AATTC 3 G G 3 CTTAA 5 Join DNA fragments with DNA ligase New DNA composed of original molecule and inserted molecule. 5 3 3 G AATTC G AATTC 5 CTTAA G CTTAA G Why do you think this is such an important procedure? If you thought about inserting DNA from one organism into another organism, making certain drugs, or fixing a genetic disease then you are on the right track. For example, if a scientist wants to study a specific protein, she can insert the gene for that protein into bacterial DNA and have millions of bacterial cells make the protein. It is not quite that simple, but you get the idea. Restriction enzymes also allow scientists to create maps of DNA based on the postion of restriction sites. One kind of physical map of DNA is a restriction map. Restriction maps include the location and order of sites cut by different restriction enzymes (see Figure 3). These physical maps are important to scientists as they identifiy and work with DNA molecules. 3
Figure 3. Restriction map of lambda DNA cut with BamHI 5505 16,841 5626 6527 7233 6770 Fragment sizes basepairs (bp) How do you create a restriction map? After the DNA is cut with different enzymes, scientists separate the fragments by gel electrophoresis and analyze the resulting patterns. Gel electrophoresis is a common way to measure molecular sizes. Here is an analogy to explain gel electrophoresis. Imagine a very dense forest of trees where each tree is only one foot away from every other tree. You and a mouse must each run through the forest from point A to point B. You and the mouse begin at the same time. Which will reach the other side first - you or the mouse? In gel electrophoresis, the gel is analogous to the overgrown forest. The gel is a mesh formed of polymers, much like JELL-O. JELL-O is a mesh of gelatin made from animal products like collagen. The gels for electrophoresis are a mesh of agarose, which is purified from seaweed. In gel electrophoresis the molecules are analogous to you and the mouse. Small molecules will move more quickly through the mesh than will large molecules, for the same reasons that the mouse moves more quickly through the thick forest than you do. So far so good, but once a molecule is suspended in the gel, why does it move? DNA, RNA and protein molecules often have a surface charge. This means that if an electric field is applied to the gel, molecules with a negative surface charge will move through the gel towards the positively charged anode and molecules with a positive surface charge will move through the gel towards the negatively charged cathode. When two (or more) molecules have the same charge the molecules will move through the gel based upon their size, smaller molecules move faster through the gel then larger molecules (Figure 4). Figure 4. Agarose gel electrophoresis of DNA http://www.discoveryandinnovation.com/biol202/notes/lecture23.html In this lab, you will being using DNA from a bacteriophage called lambda ( ) and cutting it with two enzymes: EcoRI and HindIII. Once you have cut the DNA, you will run it on an electrophoresis gel and compare lambda ( ) cut with the two different enzymes and the uncut lambda ( ) DNA. To make things a little more interesting, the enzymes are not identified. There are two tubes A and B. One has EcoRI, and the the other has HindIII. Your job is to figure out which tube contains which enzyme. 4
Molecular Scissors Pre-Lab Questions Directions: After reading through the introduction and protocol for the Molecular Scissors lab, answer the following questions. 1. What are restriction enzymes, and where are they found in nature? 2. You find a type of bacteria that is resistant to the T-4 bacteriophage. How might you explain the bacterium s resistance to this virus? 3. The restriction enzyme PvuI recognizes a 6 base pair, palindromic sequence in double stranded DNA. Three bases of one strand are given. Complete the restriction site for PvuI. 5 T C G 3 3 5 PvuI cuts both strands of DNA. The position of the first cut is indicated by the arrow above. Draw an arrow to indicate the position of the second cut. 4. Imagine you have a circular piece of bacterial DNA (a plasmid) in which you want to insert a gene to study a protein. You need to decide what restriction enzyme to use to cut open the plasmid EcoRI or HaeIII. What enzyme would you use to cut the plasmid? Explain your answer. 5. Many molecules of DNA from a virus have been cut with various combinations of the restriction enzymes EcoRI, BamHI, HindIII. The restriction map and resulting gel are shown below. Which sample on the gel shows DNA that has been cut with both BamHI and HindIII? Explain your answer. DNA from virus 1 2 3 4 wells EcoRI BamHI EcoRI HindIII 5
Molecular Scissors Lab Protocol There s been a mix up in the lab labels on your tubes of restriction enzymes have fallen off. You know that you have a tube of EcoRI and a tube of HindIII, but you don t know which one is which. In order to identify the enzymes in tubes A and B, you will have to perform digests of DNA from a bacteriophage called lambda ( ). Once you have cut the DNA you will run it on an electrophoresis gel to compare the cut and uncut DNA and the results of the two different enzymes. By comparing the gel results to the restrictions maps in Figure 5 you will be able to figure which tube contains which enzyme. Figure 5: Restriction Maps for Lambda/HindIII and Lambda/EcoRI Materials: check your workstations to make sure all supplies are present before beginning the lab. Student Workstation: Common Workstation: 1 ice bucket or styrofoam cup with crushed ice 37 C water bath or incubator 1 p20 micropipette and pipette tips 65 C water bath or incubator 1 microcentrifuge tube rack microcentrifuge (optional) 1 tube with 200 L water UV light or blue light 1 tube CutSmart buffer with 20 L buffer transilluminator 1 tube lambda DNA with 10 L DNA (500 ng/ l) 1X electrophoresis buffer 1 tube Enzyme A with 1 L restriction enzyme 1 tube Enzyme B with 1 L restriction enzyme 1 tube DNA Ladder with 10 L Quick Load 1kb Extend Ladder 1 tube Control (empty) 1 tube loading dye with 30 L 6X dye 1 agarose gel (0.6%) with DNA stain 1 electrophoresis unit with power supply 1 extra fine point permanent marker 6
Caution: Keep all reagent tubes on ice. Procedure: 1. Find the four microcentrifuge tubes labeled enz A, enz B, Control and Ladder 2. Using your p20 micropipette, set up the restriction digests using Table 1 below. It is a good idea to check off the reagents as you add them. Table 1: Set-up for restriction digest of lambda with EcoRI and HindIII. Add the Tube Following Reagents: Enz A Enz B Control Ladder Water 42 L 42 L 43 L 5 L CutSmart Buffer 5 L 5 L 5 L none DNA 2 L 2 L 2 L none Enzyme A (already added) 1 L none none none Enzyme B (already added) none 1 L none none Ladder (already added) none none none 10 L Total Volume 50 L 50 L 50 L 15 L Caution: Make sure you use a clean tip for each pipetting transfer. 3. Using the centrifuge, quickly spin the tubes to get all the reagents to the bottom. If you do not have a centrifuge, you can gently tap them on the bench to consolidate the contents in the bottom of the tube. 4. Put all tubes except the Ladder in the 37 C water bath for 30-45 minutes or for the time it takes you to make your gel. 5. During the incubation, add 5 L of sterile water to the tube labeled ladder. 6. Prepare or source a 0.6% agarose gel and 1X electrophoresis buffer as instructed by your teacher. 7. Remove your reaction tubes from the 37 C water bath. Stopping Point Check with your teacher before continuing with the protocol. 8. Add 8 L of loading dye to the reaction tubes (Enz A, Enz B and Control). Mix by flicking the tube and pool reagents at the bottom by centrifuging or by tapping the tube on the bench. Caution: Make sure you use a clean tip for each reaction tube. 9. After loading dye is added, heat your reaction tubes to 65 C for 5 minutes. Move tubes directly to ice. Caution: Do not heat the Ladder. 7
10. Assemble the gel box and position it where it will run. Tip: Check to make sure that the gel tray is in the correction orientation with the wells closest to the negative electrode. 11. Load 15 L of each sample and the ladder into a well of the gel. Record which sample went into which well. Use Table 2 to help you know where on the gel each sample is loaded. 12. Run your samples as instructed by your teachers until the front loading dye is two-thirds of the way down the gel. Table 2: Gel electrophoresis Caution: Make sure you use a clean tip for each sample. Lane (left to right) Sample name 1 2 3 4 5 6 7 8 13. While the gel is running, calculate the size of the restriction fragments that you expect when lamda DNA is cut with each of the enzymes. Record them in Table 3 below. Table 3: Single digest restriction fragments of Lambda DNA cut with EcoRI and Hindlll Restriction Fragments 23,130 2,027 HindIII Fragments in descending order Restriction Fragments EcoRI Fragments in descending order Hint: for the Hind III digest, look at the map in Figure 5. Starting at start and moving to the first restriction site, the size of the fragment is 23,130 bp. To determine the size of the second fragment, subtract the base pair number of the first site (23,130) from the number of the second site (25,157). Continue this process, recording the size of the fragments in the table. When you have finished, in the second column of the table list the fragments in descending order. 14. Once your gel is finished running, examine the location of the bands, and complete the data collection worksheet. 8
Molecular Scissors Data Collection Worksheet Directions: After completing the Molecular Scissors lab, answer the questions below. 1. On the image below draw what you see after gel electrophoresis Ladder (Kb) 48.5 20 15 electrode 10 8 6 5 4 3 2 1.5 1 0.5 +electrode 2. Determine which enzyme was EcoRI and which was HindIII. Record it below. Enzyme A Enzyme B 3. The restriction site for EcoRI is GAATTC. Based on your results, how many times does the sequence occur in the lambda sequence? 4. When you compare the restriction maps to your results, are there any missing bands? Can you suggest any reasons why there is this discrepancy? 5. What is the total length of the Lambda DNA? 9
Molecular Scissors Post-Lab Questions and Analysis Directions: After completing the Molecular Scissors lab, answer the questions below. Scientists can use electrophoresis results to determine the size of unknown DNA samples. Scientists have determined that DNA fragments move through the gel at at rate that is inversely proportional to the log 10 of their molecular weight (or number of base pairs). So, if you run a set of fragments with known sizes on a gel along with the unknown fragment, you can create a standard curve with the known fragments and use it to determine the size of the unknown fragments. Let s try it. 1 2 3 4 5 A B C 1. The gel image above depicts a gel with a DNA standard in lane 1 and two unknowns in lanes 3 and 5. Measure the distance that each band in the DNA standard has moved from the well and record it next to the corresponding band in Table 4 (on the next page). Tip: You should measure from the front edge of the well to the leading edge (furthest from the well) of each band. 2. Measure the distance that each of the unknown bands has traveled from the wells and record the distance in Table 4. 3. Use the data from Table 4 to create a standard curve using either Excel or semi-log paper. The x axis is distance traveled, and the y axis is base pair length. Connect the data points with a best-fit line. 4. Use the standard curve to determine the size of the three unknown fragments. Record the size of the fragments A, B and C in Table 4. 10
Table 4. Restriction digest gel of two unknowns. DNA Standard Size (bp) of DNA Distance (mm) moved from well 23,000 Skip this band very large bands will skew the standard curve Band 9,416 30.9 B A Size (bp) of DNA Unknowns Distance (mm) moved from well 6,557 C 4,361 2,322 2,027 5. Use the data from Table 4 to create a standard curve using either Excel or semi-log paper. The x axis is distance traveled and the y axis is base pair length. Connect the data points with a best-fit line. 6. Use the standard curve to determine the size of the three unknown fragments. Record the size of the fragments A, B and C in Table 4. 11
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Maps of Lambda DNA with EcoRI and HindIII Restriction sites 13