DNA Fingerprinting Introduction Laboratory techniques called DNA fingerprinting have been developed to identify or type an individual's DNA. One application of these techniques has been in solving crimes. Recall the way a restriction enzyme works. Remember that it is specific for a certain sequence and will cut the DNA at that sequence. DNA fingerprinting works on the premises that, while we all have the same genes, there are many different alleles for those genes and therefore the exact sequence of nucleic acids is different from individual to individual. It follows that the location of restriction enzyme sites within one person's complement of DNA is different from another person's. This section will show you how this characteristic can be taken advantage of. The DNA fingerprinting technique is summarized as follows: 1. DNA is extracted from the nuclei of a sample of white blood cells. 2. Restriction enzymes cut DNA strands into fragments called restriction fragment length polymorphs or RFLPs. 3. The mixture of RFLPs is placed in an electrophoresis gel and separated by differences in size.. RFLPs are treated with an alkaline solution to open the DNA chain and then transferred and glued to a filter. 5. The RFLP filter is then treated with a short chain, complementary radioactive DNA probe. 6. The filter with bound radioactive probe is then exposed to X ray film and a banded picture is obtained, with each band representing an RFLP combined with the probe. 7
DNA Fingerprinting: Solving a murder case with Biotechnology Homicide investigators have been called to the scene of a murder. They found blood stains of two different types on the victim's body. One of the stains was clearly the blood (type 0) of the victim, and the other was (type 0) clearly the murderer's. Four suspects have been apprehended. Each of them has type 0 blood. You and your group are forensic scientists chosen to present evidence as to which of the four suspects is the murderer. You decide to use DNA fingerprinting to accomplish your task. Follow the steps precisely as they are written. Each step in the actual procedure is witnessed, signed, and dated. A mistake could send an innocent person to prison, or result in the release of the actual criminal. Procedure 1. Work in groups. The class will need to analyze six blood samples; each group should work on one blood sample. Divide up some of the assigned tasks to speed the process, and be sure to check with one another as you proceed, to ensure that directions are being followed correctly. Obtain these materials per group: A. Four plastic with the following: 18 thymine nucleotides (orange, white and red beads) 18 adenine nucleotides (yellow, white and red beads) 18 guanine nucleotides (green, white and red beads) 18 cytosine nucleotides (blue, white and red beads) B. One plastic bag with two radioactive probes made from the following: 6 pink (radioactive phosphate), 6 white, 2 orange, 2 green and 2 blue beads C. One plastic bag with 30 plastic connectors (hydrogen bonds) D. In addition pick up the following: 2 clear plastic restriction enzyme cards, "Jan I" & "Ward II" 1 clear plastic alkali card 2. The instructor will cut up a DNA sheet that contains DNA base sequences obtained from the blood of the victim, the four suspects' blood and the murderer's blood from the crime scene. The instructor will give each group of students a strip of paper which contains the DNA base sequence from a particular person. 3. Carefully assemble your given DNA with the colored beads (nucleotides), using the given color codes and the paper DNA strip provided. In all of the strips the 5' end and three thymine nucleotides SHOULD ALWAYS BE ON THE TOP LEFT. A sample DNA molecule is in Figure 1. 75
Restriction Enzyme Card 5' 3' @) 1 - -------~ A I (!) (i) 3' 5' Jan I Using the Restriction Enzyme Card. Look at your two restriction enzyme Cards. These enzymes will make cuts in your DNA in the manner indicated by the dotted lines. Restriction enzymes used in recombinant DNA applications recognize palindromic sequences, but for the purposes of this exercise these imaginary enzymes will only examine the DNA from left to right as it is lying on the table in front of you. Begin with the restriction enzyme JAN I. Place it on top of the left side of the beaded DNA chain so its label is right side up. Move the card along the surface of the DNA until you match the precise sequence shown on the card. Stop and break the beads apart in the manner indicated by the dotted lines. The break should be in such a manner that all of the resulting strands still have the appropriate beads to represent 5' and 3' ends. Move the enzyme card until you reach the right end of the DNA. It is a good idea to double check to ensure you have made all the possible cuts. Repeat the procedure on the resulting DNA fragments using the WARD II restriction enzyme. Be sure to keep the DNA fragments in the orientation described previously (5' orange thymine beads on top left) throughout the procedure. You should now have several DNA fragments of different lengths with sticky ends. In the actual lab procedure, restriction enzyme digestion of DNA results in millions of DNA fragments, each one thousands of nucleotides long. 76
Gel Runs or Columns DNA Fragments ~ ' : ' j I I -- ' ' In actual laboratory practice, the molecules are not visible. The radioactive probes will expose a photographic plate and leave an image of their position on the gel. 5. The next step is to separate these fragments from each other by size. This is accomplished by exposing the RFLPs to a process called gel electrophoresis. Look at the enlarged paper Gel Electrophoresis Lane Sheet on the next page. Notice that the lane has a negative end and a positive end. DNA has a negative electrical charge, so the RFLPs are attracted to the positive pole of the gel lane when an electric current is applied. Place the RFLPs at the negative pole of the lane and simulate the migration by moving your RFLP's along the lane. Shorter fragments are lighter in weight and move faster than longer fragments. When the current is removed, the short RFLPs have moved further than the long ones. To determine the final position of the RFLPs, count the number of nucleotides on the longest side of each fragment. Place each measured RFLP next to its corresponding length marked on the gel lane. For each RFLP, make a small mark on the left side of the gel lane, next to the length printed on the gel lane sheet. In the actual procedure, gel electrophoresis may take hours to complete. Have your instructor check your RFLP pattern distribution on the paper gel electrophoresis lane. At this point we have successfully separated the RFLPs of all the subjects and you could walk around to all of your classmates 'gels' and figure out the identity of the murderer. However, remember that these are tiny, tiny molecules and can't be seen with the naked eye or any but the most powerful electron microscope. We will have to use an imaging process. An imaging process involves attaching something we can see to something we can't see. In our process 77
we are going to attach a radioactive probe to the RFLPs. Since we can detect the radioactivity of the probes, we will be able to pinpoint the location of the RFLPs. 6. Treatment with an alkali substance breaks the hydrogen bonds holding the two strands of DNA together causing them to unzip and become single-stranded. Obtain an alkali card and pass this card over each of the RFLP's. Remove the hydrogen bonds that hold the nucleotides on complementary strands together to produce single-stranded RFLPs. At this point in the actual lab procedure, single-stranded RFLPs are transferred and chemically glued to a rigid filter. The filter is easier to work with than the wobbly gel. 7. A cdna probe is a relatively short (usually about 8-20 nucleotides long) piece of single-stranded DNA that is designed to find and stick (hybridize) to its complementary sequence in one or more of the RFLPs. Our probe is only three nucleotides in length. Remove the radioactive probes ( cdna) from the plastic bags provided. In the actual lab procedure, millions of copies of the probes are used. The probe copies are exposed to the RFLPs on the filter. Now remove the lower single-stranded RFLPs. Search all your upper single-stranded RFLPs for the complementary sequence to the cdna probe and attach a cdna probe to any of the appropriate sequences. Radioactive Probe R = radioactive phophate = pink bead 8. The next step is to detect the radioactive probes. The principle behind this is the same as is used when you get an X-ray, when a stream of mild radioactivity is directed through your body. When it hits a film behind you it exposes the film. Where there are bones in your body the stream of radioactivity is interrupted and the corresponding piece of film is not exposed. This results in the familiar patterns of X-rays. By placing a piece of film over the filter with the RFLPs the radioactive probes will expose the portions of the film to which they are immediately adjacent. This results in a banding pattern called an autoradiograph. On your Gel Electrophoresis Lane, draw a dark line horizontally across the gel at the number of nucleotides in the fragments to which the radioactive probes are attached. 78
Using the Student Gel Lane Sheet, sketch dark bands at the correct positions in the gel lane reserved for your DNA sample. Transfer your results to the Student Analysis Sheet for your suspect. Mark all the suspect results from the other teams on the same sheet. The Student Analysis Sheet simulates what would be used in the actual lab procedure. In the actual procedure, a single autoradiograph would contain banding patterns for all six (victim, suspects, and murderer RFLP/cDNA probe bands) lanes. The instructor will identify the team who was assigned the murderer's DNA. 9. When the instructor has confirmed your conclusion, return the entire assembled radioactive probes back to their bags, and disassemble and return the DNA fragments as nucleotides to the appropriate containers! Recall that the white _bead's connection knob should be projecting freely for each nucleotide trio. 79
Student Gel Lane Sheet I 22 21 I 20 l 19 I la I 17 I 16 I 15 1 13 12 l l 10 9 -- 8 7 6 5 3 2 l J I Name Date Suspect No. --- 80
00 - Victim's Suspect Suspect Suspect Blood 1 2 3 _ 22] 21 I 20 I 19 1 18 17 \ 16 I 1s I 1 I 13 1 12 I 11 I 10 I 9 I 8 7 6 5 3 2 1 ~ 22 t- 21 1-- 20 f-- 19 : ~~ r-- 16 1-- ~ ~~ ~-- 13 l---- 12 ~~! f 7 : 6! - ~ 1-3 ; ~ 22 21 20-19 18 1 17 I 16 1 15 1-13 I 12 I 11 I 10 I 9 I 8 I 1 I - 6 I 5 I I 3 I 2 I 1 Position of RFLPs 22 21 20 19 18 17 16 15 1 13 12 11 10 9 1-8 7 6 5 3 1-2 1-- 1 Suspect r--- r-- 22 ~ 21 r- 20 ~ 19 f-- 18!. 17 ~ 16 15 1 I 13 1 12 11 10 9 8 7 i- -- 6 5 3 2 1 Murderer 221 21 20 19 18 17 I 16 I 15 I 1 13 12 11 10 9 8 7 6 5 3 2 1
Questions: 1. What is the principle underlying the DNA fingerprinting method? 2. Which of the suspects is the real murderer? Explain your answer. 3. What features of your beaded DNA segment in the fingerprinting exercise are not shown on the paper representations of DNA segments?. Will the probes hybridize to the same RFLPs in all five suspects' RFLPs? Explain your answer. 5. What advantage would you have if you used a combination of different probes? 6. Why is alkali used before the cdna step? 7. Radioactivity trapped on the RFLP filter is most likely due to what effect? 8. What is the role of autoradiography in DNA fingerprinting? 82
9. What do you think is more important in DNA fingerprint analysis: the number of fragments or the position of the fragments on the autoradiograph? Explain your answer. 10. How could you prove to ajury that DNA fingerprinting works? 11. Sometimes the fingerprint pattern is incomplete because certain RFLPs run off the gel (although their path can be seen with a tracking dye.) Which RFLPs (large or small) are potentially lost in the fingerprinting technique? 12. State possible limitations to DNA fingerprinting, i.e., what steps in the technique itself might produce invalid or incomplete results? 13. Besides solving crunes, what other benefits could be associated with DNA fingerprinting? 1 This exercise has been adapted from the exercises developed by the Biology Department at San Diego City College and used by permission. 83