Forensic DNA Fingerprinting: Using Restriction Enzymes Bio-Rad DNA Fingerprinting Kit Background: Scientists working in forensic labs are often asked to perform DNA profiling or fingerprinting to analyze evidence in law enforcement, mass disasters, and paternity cases. In this laboratory activity, you will enter into the role of a forensic scientist who has been called upon to help solve a crime. You will use forensic techniques, and the first steps will be to gather DNA found at the crime scene and obtain DNA samples from five suspects. The DNA will be digested with a fixed set of restriction enzymes, separated on a gel by gel electrophoresis, and then analyzed for patterns of similarity with the crime scene sample. From these results, you will make recommendation to identify the perpetrator. Restriction enzymes are a special class of proteins that cut DNA at specific sites and have become an indispensable tool in molecular biology. Restriction enzymes, also known as endonucleases, recognize specific sequences of DNA base pairs and cut, or chemically separate, DNA at that specific arrangement of base pairs. The specific sequence of DNA is called a restriction site. These unique enzymes occur naturally in some bacteria and act to protect them from invading viruses. Viruses called bacteriophages, phages for short, attack bacteria by inserting their genetic material into the bacterial cell. The phage commandeers the bacterial cell, replicating rapidly until the bacterial cell lyses and releases more phages to carry out the same infection process in neighboring cells. However, if the bacterial strain has restriction enzymes that recognize restriction sites on the invading phage nucleic acid, then enzymes can destroy the invading genetic material by digesting and inactivating the phage genes. Bacterial cells protect their own DNA from being self-digested by modifying certain nitrogen bases along their genome, this prevents their restriction enzymes from recognizing and digesting their own sequences. Restriction Enzymes as Molecular Scissors: Scientists use restriction enzymes as tools to assist with DNA splicing. Restriction enzymes are used to cut both the DNA sequence of interest and the host DNA; they act like molecular scissors. Their discovery led to the development of recombinant DNA technology that allowed, for example, the large scale production of human insulin for diabetics using E. coli bacteria. Many restriction enzymes have been studied in detail, and more than 600 are available commercially and are used routinely for DNA modification and manipulation in laboratories. Gel Electrophoresis Once the DNA has been digested, the soup of fragments must be separated in order to learn more about each fragment, such as its. Gel electrophoresis is a method used to separate DNA fragments based on their s by applying an electrical field to an agarose gel containing these DNA fragments. DNA contains many negative electrical charges, and scientists used this property to separate pieces of DNA. Once the fragments are loaded onto the gel, an electrical current is applied causing the negatively-charged DNA molecules to move towards the positive electrode (red). The agarose gel acts as a matrix of tiny pores that allow small particles to move through it relatively quickly. The larger fragments migrate much more slowly through the gel. After a set period of exposure to the electrical current, the DNA fragments are separated by with the smaller ones located further away from the wells than the larger fragments. 1
Fragments that are either the same or very similar in will tend to migrate together through the gel. Fragment bands form as a result of the various distances the DNA segments migrate. DNA Fingerprinting Your mission begins with the collection and restriction digestion of Crime Scene and Suspect DNA. When DNA is mixed with restriction enzymes, the enzymes act as molecular scissors and digest the DNA into smaller pieces. However, these cuts are not random. The enzymes look for specific DNA sequences, restriction sites, and make specific cuts at those locations. The resulting fragment s are dependent on how often the restriction sites occur within the DNA. This means that identical DNA sequences will produce identical DNA fragments when the DNA is digested with the same restriction enzymes and different DNA sequences will produce different d fragments when digested with the same restriction enzymes. Your task will be to separate the DNA fragments based on using a procedure known as gel electrophoresis and then to compare the DNA fragments with those of a standard ladder whose base pair s are already known. Molecular Weight Standards (also known as Markers or Ladders): Being the professional forensic scientist that you are, you will quantitate the of the DNA fragments in both the crime scene and suspects samples. To determine the s o the experimental DNA bands, they must be compared to a standard ladder that has DNA bands with known molecular weights. The standard used during activity is the HindIII digest of lambda. DNA, a commonly used DNA molecular weight marker. The standard ladder is run in a lane next to the experimental lanes under the same conditions. Linear DNA fragment migration distance through a gel is inversely proportional to the log10 of its molecular weight (base pair number). 2
Day 1 Preparing the DNA Samples Quick Guide for DNA Fingerprinting Kit - UView 1. Place the tube containing the restriction enzyme mix, labeled ENZ, on ice. 2. Find your tubes containing the DNA from the Crime Scene (CS) and 5 suspects (S1-5). 3. Add 6 ul of enzyme mix (ENZ) into the very bottom of each tube. Use a separate tip for each sample. 4. Cap the tubes and mix by gently flicking the tubes with your finger. Pulse-spin the tubes in a centrifuge to collect all the liquid in the bottom of the tube. If a centrifuge isn t available, tap the tube on a table top. 5. Place the tubes in the floating rack and incubate for 45 minutes at 37 C, or overnight at room temperature in a large volume of water heated to 37 C. 6. After the incubation period, remove the tubes from the water bath and place in the refrigerator until the next laboratory period. 3
Day 2 Gel Electrophoresis Remove your digested DNA samples from the refrigerator. If a centrifuge is available, pulse spin the tubes in the centrifuge to bring all of the liquid into the bottom of the tube. 1. Using a separate tip for each sample, add 3 µl of loading dye "LD" into each tube. Cap the tubes and mix by gently flicking the tube with your finger. 2. Place an agarose gel in the electrophoresis apparatus. Fill the electrophoresis chamber with 0.25x TAE buffer to cover the gel, using approximately 275 ml of buffer. 3. Check that the wells of the agarose gels are near the black (-) electrode and the base of the gel is near the red (+) electrode. 4. Using a separate tip for each sample, load the indicated volume of each sample into 7 wells of the gel in the following order: Lane 1: M DNA marker 15 µl Lane 2: CS green 15 µl Lane 3: S1 blue 15 µl Lane 4: S2 orange 15 µl Lane 5: S3 violet 15 µl Lane 6: S4 red 15 µl Lane 7: S5 yellow 15 µl 5. Place the lid on the electrophoresis chamber. The lid will attach to the base in only one orientation. The red and black jacks on the lid will match with the red and black jacks on the base. Plug the electrodes into the power supply. 6. Turn on the power and electrophorese your samples at 200V for 20 minutes. 7. When the electrophoresis is complete, turn off the power and remove the top of the gel box. Carefully remove the gel and tray from the gel box. Be careful the gel is very slippery! 8. View using a UV Transilluminator or UV lamp. 4
Results: Take a picture of your Final Gel for your Deliverable Lambda/HindIII marker Band Actual Crime Scene Suspect 1 Suspect 2 Suspect 3 Suspect 4 Suspect 5 1 23,130 2 9,416 3 6,557 4 4,361 5 2,322 6 2,027 Quantitative Analysis of DNA Fragment Sizes: 1. Using a ruler, measure the distance (in mm) that each of your DNA fragments or bands traveled from the well. Measure the distance from the bottom of the well to the center of each DNA back and record your numbers in the table above. The data in the table will be used to construct a standard curve and to estimate the s of the crime scene and suspect restriction fragments. 2. To make an accurate estimate of the fragment s for either the crime scene or suspect DNA samples, a standard curve is created using the distance (x-axis) and fragment (y-axis) data from the known Hind III lambda digest (DNA marker). Using both linear and semi log graph paper, plot distance versus for bands 2-6. On each graph, draw a line of best fit though the points. Extend the line all the way to the right hand edge of the graph. 3. Decide which graph, linear or semi log, should be used to estimate the DNA fragment s of the crime scene and suspects. Justify your selection. 4. To estimate the of an unknown crime scene or suspect fragment, find the distance that fragment traveled. Locate that distance on the x-axis of your standard graph. From that position on the x-axis, read up to the standard line, and then follow the graph line over to the y-axis. Where the graph line meets the y-axis, this is the approximate of your unknown DNA fragment. Do this for all crime scene and suspect fragments. 5. Compare the fragment s of the suspects and the crime scene. 5
Graphs: Linear Paper Semi log Paper Analysis Questions: 1. Do any of your suspect samples appear to have EcoRI or PstI recognition sites at the same location as the DNA from the crime scene? If so which ones? 2. Based on the above analysis, do any of the suspect samples of DNA seem to be from the same individual as the DNA from the crime scene? Describe the scientific evidence (be sure to discuss base pair s) that supports your conclusion. 3. What are restriction enzymes? How do they work? What are recognition sites? 4. What is the source of restriction enzymes? What is their function in nature? 5. Describe the function 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. A certain restriction enzyme digest results in DNA fragments of the following s: 4,000 base pairs, 2,500 base pairs, 2,000 base pairs, 400 base pairs. Sketch the resulting separation by electrophoresis. Show starting point, positive and negative electrodes, and the resulting bands. WHAT YOU WILL TURN IN: THE DELIVERABLE Your final product for this lab will be turned in as a group of TWO via google classroom and include the following: 1. Picture of your final gel 2. Table of results 3. Linear graph & Semi Log graph a. Justify your selection of the linear or semi log graph in estimating the DNA fragment of the crime scene and suspects. 4. Analysis Questions You will submit your work as a google slide in Google Classroom. Be sure to include BOTH members names. 6