The Science Detectives- Murder in a Science Lab

Transcription

1 CASE STuDY- Evidence Docket Crime Scene Investigation- Scenario: You and your Crime Scene Investigation Unit arrive on the scene of a crime. A man named Dr. Darren Hobbs was found lying on the floor of his office, adjacent to his laboratory, lifeless. A handwritten suicide note lays hastily scrawled on the desk beside where Dr. Hobbs was found, but is immediately discounted as being forged and is not believed to be written by the deceased. Given the position of the body, amount of physical evidence, and falsified suicide note, we suspect that Dr. Hobbs was a victim of foul play and his death appears to be a homicide. Although Dr. Hobbs was well respected and well liked someone must have wanted him dead. The electronic door access codes have identified 2 suspects that entered and exited the lab between when Mr. Hobbs was last seen alive and when his body was discovered. Suspect1- Clara Flowers- Lab Assistant, Clara has worked in research labs for the last 10 years. She came to work with Mr. Hobbs 5 years ago. She helps prepare solutions and manages inventory of lab supplies. Suspect 2- Howard Brown- Graduate Student, Howard is nearly done with the research for his doctorate. He has been Dr. Hobbs s student for the past 3 years. He is considered one of the most talented young minds in his field, a distinction one held by Dr. Hobbs. We are able to collect the following types of evidence- Chemical Evidence- The forged ransom note written in black ink, a portion of which was collected and processed by the first responders field chemical analysis team. This team has returned the initial chemical signature results obtained from the ink analysis. Fingerprint Evidence- Fingerprints from Dr. Hobbs, Suspect 1 and Suspect 2 are provided for your consideration. Physical Evidence- Each of the two pens, one belonging to each suspect was collected from inside Dr. Hobbs s office. We have access to the laboratory materials that Dr. Hobbs and his team had been using the day the crime was committed, please evaluate any useful materials in the evidence box. Genetic Evidence- DNA reference samples have been obtained from Dr. Hobbs and his immediate colleagues, since these genetic profiles must be stored in the laboratory. The first responders team has collected DNA from the deceased and processed assault DNA from the body, including scrapings from under Dr. Hobbs s fingernails and analysis of hair found clenched in his hand. Rapid genetic fingerprinting results from the first responders have been returned for your consideration. 1

2 Technique 1- thin Layer Chromatography Technique 1- Thin Layer Chromatography Background: A common and simple technique for distinguishing chemical compounds is Thin Layer Chromatography (TLC). TLC takes advantage of the differences in polarity of molecules. The two major components of TLC, like all chromatography, are the mobile phase and the stationary phase. The TLC plate is considered the stationary phase. It consists of a glass plate, thinly coated with silica gel (solid). Silica gel is very polar and has a high surface area, allowing for rapid absorption. Nonpolar liquids are used for the mobile phase of TLC. In this investigation we will use acetone (other examples include ethanol, turpentine, nail polish remover, paint thinner, lighter fluid). When acetone is in contact with silica gel, it moves through the silica gel matrix via capillary action. The edge of the solvent climbing the TLC plate towards the top of the plate is called the solvent front. For the diluting portion of your work we will be using ethanol to dilute your test inks from each pen. Other solvents may work, but ethanol is best for our purposes. Materials: 1 Developing jar, or 50mL plastic tube with lid 1 TLC plate (careful not to touch the white silica) 2 test pens, and 2 scraps of paper 2 2μL tubes Scissors 10μL pipette 1 graphite pencil Ethanol and acetone This is an example of a TLC plate. The line at the top is the solvent front; the spots come next, then at the very bottom are the blots. EVIDENCE- The initial analysis of a clipping from the ransom note has revealed an ink pattern that is characterized by a yellow spot with an Rf value of approximately

3 Technique 1- thin Layer Chromatography Thin Layer Chromatography Method: 1. Label your 50mL tube with the investigator or group s name. 2. Label each of the 2 2μL tubes for the appropriate suspect pen. 3. Color a small scrap of paper as darkly as you can with each pen; be sure to fully saturate the scrap of paper with ink. Don t forget to remember which scrap contains which ink. 4. Add 300μL of ethanol to each small tube, and the colored paper scrap, then screw closed. 5. Shake and agitate the sample until all the ink from the paper has diluted into the ethanol. Repeat the same for the second piece of paper if you haven t done so already. 6. Lay the TLC plate flat on the table. Use a pencil to gently draw a horizontal line on the TLC plate near the bottom. Try not to etch into the silica. This line should allow the bottom of the plate to be submerged into liquid without the liquid coming over the level of the line. 7. Label which sample will be spotted on the left side of the plate and which will be spotted on the right. 8. Use the 10μL pipette to spot ink from each mini tube. Try spotting 3μL three times for a dark ink blot. 9. Allow ink spots to dry. 10. Add acetone to the 50mL tube to just below the 5mL line. Stabilize this tube in a rack. Carefully add your TLC plate standing up with the ink blots at the bottom and cover the 50mL tube. 11. Let this sit until the solvent is near to the top of the plate, but remove plate before the solvent reaches the very top of the plate. 12. Remove the plate and immediately draw a line with a pencil where the solvent front ended, then circle the fragments. 10. Sketch the TLC plate into the observations portion of your worksheet label the blots, fragments, solvent front, blotting line and note any observations here (i.e. color and shape of fragment, distance the fragment moved from initial point). 11. Calculate your Rf values (see below) for both dots and compare to the Rf value from the false suicide note to see if we can match either suspect pen to the note. Discuss possible conclusions with your group and write them down in the provided section of this sheet. 3

4 Technique 1- thin Layer Chromatography Measuring and Calculating Rf Value: To calculate your Rf values measure from the line drawn where the blots were made up to a spot, this is you re A value. Next measure from the blot line to where the solvent front ended this will be your B value. Last divide your A value by your B value (A/B=Rf) and record your answer. This calculation is done for each blot on the TLC plate. These measurements should be drawn and recorded in the space provided on the following sheet. Using your calculated Rf values compare the two samples to the unknown ink from the ransom note. Observations: Sketch and label your TLC plate here. Record written observations about the appearance of the plate, spots, any differences, as well as all relevant measurements Appearance of each sample Any differences? A 1 =, B 1 = Rf 1 = A 2 =, B 2 = Rf 2 = Conclusions: 4

5 Technique 2- Fingerprint Analysis Technique 2- Latent Fingerprint Lifting Background: As early as the 14 th century people have been using fingerprints as signatures, to identify themselves. However, the first use of fingerprints to solve a crime was in a fiction novel Life on the Mississippi by author Mark Twain in Starting in 1888, Sir Francis Dalton began studying fingerprints as a means of identification; he later published the book Fingerprints, establishing the first classification system for fingerprints. This system was first used to solve a crime in 1892 when Juan Vucetich solved a murder of two brothers by their mother. Since then Fingerprinting has expanded to be commonly used at crime scenes, with the FBI even using computers to analyze several different points of identification to match fingerprints. Today the largest Automated Fingerprint Identification System collection is owned by the United States Department of Homeland Security containing over 100 million person s fingerprints. Materials: 3 reference prints Latent fingerprinting power Brushes Lifting Tape (clear tape may be used as an alternative) Evidence kit from the crime scene o Petri dish o Tube o 100 ml Pyrex bottle o PCR plate o Syringe Computer Print reference handouts. (See Appendix for source) Method: 1. On a computer complete the exercise in fingerprint pattern analysis 2. Analyze the three reference prints and fill out worksheet on the pattern and ridge characteristics 3. Observe objects and determine where fingerprints may be found. 4. Dip the brush in the fingerprinting powder and lightly brush the surface of the object. 5. Examine brushed object for prints, and carefully lift the latent print using the lifting tape provided 6. Place the tape on the worksheet. 7. Analyze the prints for print classification and ridge characteristics. 8. Repeat for each object. 5

6 Technique 2- Fingerprint Analysis Finger Printing Activity Worksheet Name: Date: Latent Print # Collected (off what surface?) How Collected (kind of powder, lift technique, etc) Classification (loop, whorl or arch) Ridge Characteristics Possible match to reference sample (and why?) Latent Print # Collected (off what surface?) How Collected (kind of powder, lift technique, etc) Classification (loop, whorl or arch) Ridge Characteristics Possible match to reference sample (and why?) Latent Print # Collected (off what surface?) How Collected (kind of powder, lift technique, etc) Classification (loop, whorl or arch) Ridge Characteristics Possible match to reference sample (and why?) 6

7 Technique 3- DNA Fingerprint Analysis Background: Although scientists have long known that DNA was our genetic material it wasn t until the 1980s when DNA was used in forensic science. Prior to this time, blood typing was the only genetic identification tool used. What problems would exclusively using blood types to investigate a crime present? The first widely used genetic tool was RFLP markers, or Restriction Fragment Length Polymorphisms. The FBI adopted using these markers in the late 1980s and stopped running RFLP markers in the year 2000 due to their cost ineffectiveness, limitations in interpretation of DNA mixtures, and difficulty multiplexing to get more than one data point at a time. Current Applications: DNA Fingerprinting by Short Tandem Repeat STR analysis is currently the standard in forensic identification. All individuals have a unique genetic profile, of autosomal STRs, besides identical twins. The FBI currently tests 13 autosomal STR loci and Amelogenin, which can detect the 2 sex chromosomes in a sample. The 13 autosomal loci are uploaded and compared to a national database called the Combined DNA Indexing System, CODIS. What are we looking at? An individual s genetic profile is unique to them. It is made up of a set of allelic scores which are heritable regions from each parent which vary slightly from person to person. We are looking for sequences that contain mutations that are short repeated series of bases, for example a region in your DNA that says CAGCAGCAGCAGCAGCAG. We call this a Short Tandem repeat, or STR. Lets look at how these appear in the DNA. Short Tandem Repeats (STRs) CAGCAGCAGCAGCAGCAGCAG--- # Copies Repeat motif= pattern of repeated bases, CAG Non- repetitive DNA = # Copies= how many times the repeat motif appears in tandem, also called allelic value 7

8 Technique 3- DNA Fingerprint Analysis Interpretation of Data- Since we know the location of and the motifs of each of these STRs we simply need to measure the length of repeated units to determine allelic value. To perform forensic identification it is necessary to examine areas of DNA useful for exclusive identification. For this purpose we test the autosomes, or non-sex chromosomes. Normal humans have 46 chromosomes, 2 of which are sex determining and 44 of which are autosomes. Each of your autosomes is organized into chromosome pairs, and one of each pair came from each parent. This means that for any given STR locus, two allelic values will be detected, one from each chromosome. Do you think that these values are the same? Sometimes, always, or never? Inheritance of autosomal alleles- The number refers to the number of times the motif is repeated. Allele 13 Allele 16 DAD Allele 10 Allele 16 CHILD Allele 10 Allele 14 MOM This diagram shows the inheritance of alleles at 1 autosomal locus. Each parent passes on one copy to their offspring. The peaks represent the repeated units fluorescing as they move through a machine, past a laser. The smaller fragments are always to the left of longer fragments. 8

9 Technique 3- DNA Fingerprint Analysis What does real forensic data look like? Locus D8S1179 has 13 repeats The image to the left shows the Identifiler reaction, which includes all 13 CODIS loci, Amelogenin to determine gender, and 2 additional autosomal markers. At any given locus where only one peak appears we interpret that score as two of the same allelic values, since the peaks are actually sitting exactly on top of one another. This is how a homozygous allelic score would appear. Next, record the allelic values for each of the three reference samples and the two evidence samples. The first one has been completed as an example. If there are two peaks at a locus, record each value. If only one peak is visible, the allelic value for each allele is identical. Dr. Hobbs- Locus (black lane) D19S433 vwa TPOX D18S51 14,14 17,19 8,10 13,18 Locus (Red Lane) AMEL D5S818 FGA X,Y 12, , 24 9

10 Technique 3- DNA Fingerprint Analysis Complete the following scores tables Clara Locus (black lane) D19S433 vwa TPOX D18S51 Locus (Red Lane) AMEL D5S818 FGA Howard Locus (black lane) D19S433 vwa TPOX D18S51 Locus (Red Lane) AMEL D5S818 FGA 10

11 Technique 3- DNA Fingerprint Analysis Fingernail scraping evidence Locus (black lane) D19S433 vwa TPOX D18S51 Locus (Red Lane) AMEL D5S818 FGA Hair evidence Locus (black lane) D19S433 vwa TPOX D18S51 Locus (Red Lane) AMEL D5S818 FGA 11

12 Technique 3- DNA Fingerprint Analysis DNA Fingerprinting Observations- Conclusions- Putting it all together- From the Chromatography were you able to identify the author of the suicide note by the ink in their pen? Given the latent prints that you lifted, was anyone other than Dr. Hobbs touching the lab supplies? Does the DNA evidence corroborate the other types of evidence, why or why not? Who do you think killed Dr. Hobbs? 12

13 DNA VOCABULARLY- Appendix 1- Vocabulary Allele- A variant form of a gene or genetic marker Autosome-Any nuclear chromosome other than a sex-determining chromosome; a chromosome that is neither an X nor a Y chromosome Chromosome- Bundles of tightly coiled DNA. Most human cells (not egg and sperm cells) have 23 chromosome pairs (22 pairs of autosomes and one pair of sex chromosomes) contained within the nucleus. One member of each pair is inherited from the mother, the other from the father. Diploid- Cell having two copies of each chromosome Gene- Region of DNA known to code for a function Genetic Marker- A DNA sequence known to have a variable site, but not necessarily a gene Haploid- Cell having a single copy of each chromosome Heterozygous- Characterized by two different alleles for the same gene or marker Homozygous- Having two identical alleles of a given gene or marker Locus- (plural loci) Location of a genetic marker or gene within the DNA Recombination- The process by which a strand of genetic material is broken and then joined to a different DNA molecule. In eukaryotes recombination commonly occurs during meiosis as chromosomal crossover between paired chromosomes. This process leads to offspring having different combinations of genes from their parents Short Tandem Repeat-STR- A genetic marker which is a repeating motif of DNA such as (CAT)(CAT)(CAT). These occur in succession and can be counted to determine an allelic value. They are particularly useful in forensic applications. X and Y Chromosomes- Chromosomes that determine gender. Females have two X chromosomes while males have one X and one Y. Since the Y Chromosome is unmatched (singular), it does not recombine with the X and is passed down intact from father to son. 13

14 Appendix 2- For teachers Technique 1- TLC- This same technique can be done using a variety of solvents. In real forensic labs chemical analysis is usually done by gas chromatography or mass spectroscopy. Flexible plates backed in aluminum and polyester are available, and offer the versatility of cut your own size, and are not as sharp as glass backed. Technique 2- Latent prints- Instead of latent print powder you can use cornstarch or graphite powder-available as lock lubricant from most hardware stores. Plain packaging tape can be used for collection of prints from surfaces. For matching by eye, a single finger comparison and analysis is the only practical way to go. In real forensic applications, any portion of the hand and palm may be lifted as a latent print, and orientation may be hard to detect. Computers are used to compare the many details of a single latent print to large fingerprint databases. We created our own reference print sheet so that the reference prints matched the evidence. This can be done using the ink form a ball point pen, or a stamp pad. Good fingerprinting references: For the history of fingerprinting: The History of Fingerprints For ridge characteristics and more information on fingerprinting: The Thin Blue Line Technique 3- DNA fingerprinting- For more information about forensic DNA typing with STRs visit the National Institute of Standards and Technology NIST at or the FBI site for more information about CODIS at To learn about the first murder conviction using DNA read up on Colin Pitchfork, who was convicted in 1988 for the 1983 and 1986 murders of two teenage girls in Leicestershire, England. This portion of the activity uses images of real forensic data. If a more hands on lab is desired try an electrophoresis gel of different sized bands. Supplies and support for this type of lab can be obtained by contacting Nadja Anderson at the BIOTECH project- Suppliers- Forensic Source- Latent print powder, $19.71 for 8oz Zephyr Fingerprint brushes $9.31 each Author Information- This activity created by Barbara Fransway at the Human Origins Genotyping Laboratory. For more information about our lab, projects and educational outreach please visit our website at Barbara can be contacted at bbf@ .arizona.edu. This activity is intended for educational use and is free for distribution to educators and students. 14