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  1. Use of Immunology Techniques in Crime Scene Analysisby Forensic Scientist Nichole Kellerman Walter Johnson High School Bethesda, Maryland High School Teachers Research Program Funded by the American Association of Immunologists

  2. Lesson 1-4 Objectives: • Review how each human has unique characteristic traits found in their DNA as genes. • Describe how Forensic Technicians use these principles to identify and exonerate suspects identified by criminal investigations. • Obtain hair samples to extract DNA. • Perform DNA extraction techniques. • Perform polymerase chain reaction (PCR). • Analyze samples with gel electrophoresis to see if PCR was successful.

  3. DNA Analysis • Each human has unique characteristic traits found in their DNA as genes. • Each cell in your body has DNA--the exception being RBC’s which mature and lose their nucleus over time. • Three types of forensic analysis of DNA are conducted: • Nuclear DNA • Y chromosomal DNA • http://projects.nfstc.org/fse/01/01-08c.html • Mitochondrial DNA • http://projects.nfstc.org/fse/01/01-08b.html • DNA analysis is divided into four parts: • Extraction • Amplification • Detection • Interpretation

  4. DNA Analysis: Extraction • DNA Extraction: • Lyse cells: Sonicating, bead beating the sample, or vortexing the sample with phenol . . . and then adding SDS, a detergent, helps to remove the lipid membranes; are effective ways for breaking apart cell membranes to release DNA from the cell. • Protease is used to degrade proteins associated with the DNA or other cellular proteins. After ammonium or sodium acetate is added to the container, a precipitate of the cells proteins will form at the bottom of the container. Then phenol-chloroform is added, and the tube is vortexed. Lastly, the conical tube is centrifuged. • The proteins will remain in the organic phase, and this makes it easier to be pipetted out of the container. • The DNA will be found at the interphase between the two phases. http://quicktubes.com/english/company/business-development/looxster.html

  5. DNA Analysis: Extraction • Add cold ethanol or isopropanol into the tube and then centrifuge. DNA will not dissolve in alcohol, so it will be seen clearly in the solution. The alcohol also acts like a washing agent, it will remove the salt that was added to the solution and keep the DNA separate from everything else. • Wash the resultant DNA pellet with cold alcohol again and centrifuge the tube again to rinse the DNA a second time. The precipitated pellet is the DNA. • Pour the alcohol off the DNA pellet. Then, re-suspend the DNA in Tris (TE) Buffer. • In order to confirm the presence of DNA, run samples in a gel. Use gel electrophoresis containing ethidium bromide or another fluorescent dye that reacts with the DNA. Place the gel under a UV light to illuminate the DNA samples. http://serc.carleton.edu/details/images/7864.html http://serc.carleton.edu/details/images/7865.html

  6. DNA Analysis: Extraction • If a sexual assault case involves processing DNA from a stain of semen, additional steps are required to separate sperm cells from the other body cells contained within the stain. The first portion of the sample is composed of sperm DNA. The other portion of the sample will contain other cells. This technique makes it possible to separate male and female DNA. • http://projects.nfstc.org/fse/01/01-09b.html

  7. DNA Analysis: Amplification • In Forensics most DNA is found in residues or stains left behind. The trace amounts of DNA can be amplified for further analysis. • Polymerase chain reaction (PCR) is a method that generates multiple copies of DNA segments or genes from a sample. Under controlled conditions, small segments of DNA are created by enzymes called DNA polymerases. The polymerases add complimentary DNA nucleotides to the existing DNA strand called the DNA template. • Smaller fragments of DNA called primers are used by the polymerase to start the process of amplifying the DNA. Primers are usually small, manmade pieces of DNA called oligomers. The primers are usually 15-20 nucleotide bases long.

  8. DNA Analysis: Amplification • During PCR, the DNA being sequenced is: • Denatured:It is heated, and the double strand of DNA is separate. • Annealed: The DNA is cooled. This allows the free floating primer sequences to bind to the DNA template strands. • Extended: The Taq polymerase attaches to the primer and uses the free floating nucleotides (ATP, CTP, GTP, TTP) to replicate the DNA sequence from the DNA template. • The number of PCR cycles (repeats) run is based on the amount of DNA desired.

  9. DNA Analysis: Detection • PCR is an invaluable tool in law enforcement as a step in the process of DNA fingerprinting. • Detection is an automated process where alleles for each gene location or locus are determined. The combinations of alleles at each locus establishes a genetic profile described by a series of letters or numbers. • Each sample is run through gel electrophoresis to determine a DNA fingerprint, a pattern of DNA fragments that are unique to each individual. http://www.2classnotes.com/digital_notes.asp?p=DNA_Fingerprinting

  10. DNA Analysis: Interpretation http://projects.nfstc.org/fse/01/01-12b.html

  11. DNA Analysis: Interpretation http://projects.nfstc.org/fse/01/01-13b.html

  12. Practical Applications of DNA Fingerprinting 1. Solving Cases of Disputed Paternity and Maternity: Because a person inherits his or her VNTRs from his or her parents, VNTR patterns can be used to establish paternity and maternity. 2. Criminal Identification and Forensics: DNA isolated from blood, hair, skin cells, or other genetic evidence left at the scene of a crime can be compared through VNTR patterns with the DNA of a criminal suspect to determine guilt or innocence. 3. Personal Identification: The notion of using DNA fingerprints as a sort of genetic bar code to identify individuals has been discussed, but this is not likely to happen anytime in the near future. The technology required to isolate, keep on file, and then analyze millions of very specified VNTR patterns is both expensive and impractical.

  13. Forensic Technicians use immunology principles to identify and exonerate suspects identified by criminal investigations. • CSI’s collect specimens left behind by the suspect or victim at a crime scene. • body fluids: blood or semen • other tissues: hair or bone • Immunological Tests are used by CSI’s in the detection and identification of crime scene samples. They perform presumptive test or screening tests at the crime scene and at the lab further analysis and confirmatory tests are completed. • DNA Typing is used to identify the collected sample against a reference sample from a known source. The reference sample could be taken from a potential suspect or a known victim. The goal is to identify the source as belonging to the suspect and not to the victim. DNA Typing can also exclude a person from a list of potential suspects. http://www.nfstc.org/pdi/Subject02/pdi_s02_m02_01.htm

  14. Explain the immunology behind confirmatory testwhen identifying bodily fluid • It is important to determine very quickly if fluid residues are blood. • Presumptive tests use a chemical reaction, linked to a color indicator, to screen the fluid sample as blood. It does not identify the origin of the blood as human or other. • A confirmatory test is needed to identifythe specific body fluid as being human blood.

  15. SerologyThe branch of laboratory medicine that studies blood serum for evidence of infection by evaluating antibody reactions in vitro. • Blood is a fluid (suspension of cells) containing 3 types of materials: • Salts: Na+, K+, & Cl- • Organic chemicals: glucose, hormones, and vitamins • Proteins • Blood contains 3 cellular components: • RBC’s or erythrocytes: • Mature circulating RBC are enucleated (lose their nucleus to increase oxygen binding ability, do not contain DNA) • Platelets or thrombocytes • Contain a nucleus with DNA • WBC’s or leukocytes • Lymphocytes are the WBC responsible for antibody production

  16. SerologyThe branch of laboratory medicine that studies blood serum for evidence of infection by evaluating antibody reactions in vitro. • Serum is the fluid portion of clotted blood: • The fluid portion of the unclotted blood is called plasma. Blood clots through the conversion of a dissolved protein, fibrinogen, to a precipitated polymer called fibrin. • Fibrin acts like a fisherman’s net, trapping platelets to form a clot. • Serum can be separated by electrophoresis into albumin andglobulin. • Albumin preserves blood volume by regulating osmotic pressure. • Globulin consists of many different proteins.

  17. Screening tests are presumptive tests • Depend on the peroxidase activity of hemoglobin, a protein within the blood that binds oxygen. • Most tests depend on the oxidation, or loss of electron, of colorless solution which is reduced, gains an electron. • Many of these solutions are conjugated to a known or suspected carcinogen. • Screening tests are not specific for blood because other organisms like fruit contain peroxidase, or it can be found on the surface of objects. • Presumptive tests can also be used to identify other body fluids: saliva, semen, vaginal fluid. . . • Preliminary tests are less time consuming, but are not definitive. Confirmatory tests are discriminating test, because they report the origin of the fluid or body tissue with a high degree of scientific certainty. They are usually performed after presumptive test because they require more time. http://projects.nfstc.org/fse/01/01-07.html

  18. Immunology behind confirmatory testused by CSI’s to identify drugs • Presumptive or Screening tests: • Determine the possible presence of controlled substances. • Classify these controlled substances into general categories: • Opium alkaloids, synthetic opiates, cocaine, indole alkaloids, benzodiazepines, barbiturates, sedatives, hypnotic, anesthetics, marijuana, and phenalkylamines • Confirmatory Tests: • Identify the identity of the controlled substance.

  19. Screening Process • Examine evidence. • Determine the location of biological evidence. • Methods used to locate stains are dependent on the type of body fluid/tissue present. • Simplest form is a swab • Little effort is required to locate the stain on a swab • Complicated evidence collection occurs with: blood on a high surface, like a window, stains on clothing, bed sheets, blankets, and carpet • These types of evidence will require more time • Many times, there will be multiple types of evidence at the crime scene. The screening process may simply be seen with visual examination, or it may require microscopes, and alternative light sources or chemical tests. http://projects.nfstc.org/fse/01/01-05.html

  20. Confirmatory Tests • Confirm the residue left behind at the crime scene contains blood. • Takayama test also known as the hemochromgen test: • The oldest confirmatory test of blood detects the presence of hemoglobin or derivatives crystals of hemoglobin. In this test, ferrous iron within hemoglobin reacts with pyridine. If red feathery crystals are present, the residue contains blood. • Teichman reagent: • In this reaction, hemoglobin is converted to hemin, and then, the halides reacts with the hemin to form brownish-yellow rhomboid crystals if the substance contains blood.

  21. Identifying Blood as being Human Blood • The next step will confirm that blood belongs to a human. • A precipitin reaction with antibodies to blood serum is used to determine that the sample is of human origin. • It is an anti-human serum, an antiserum to human serum. • In blood, serum are the proteins albumin and globulins. • The Kastle Meyer test is a confirmatory test for human blood. • http://www.youtube.com/watch?v=6Ex0Fd_PDhU

  22. Describe the basic components of the Immune system The lymph node contains numerous specialized structures. T cells concentrate in the paracortex, B cells in and around the germinal centers, and plasma cells in the medulla. Immune cells and foreign particles enter the lymph nodes via incoming lymphatic vessels or the lymph nodes’ tiny blood vessels. http://www.niaid.nih.gov/topics/immunesystem/Pages/structure.aspx

  23. Investigate the crime scene • Use the scientific method to answer these questions: • What would you consider evidence? • How would you collect, store, and document the evidence recovered? • What questions need to be answered to solve the crime?

  24. Lesson 2-5 Objectives: • Obtain hair samples to extract DNA. • Perform DNA extraction techniques. • Perform polymerase chain reaction. • Analyze samples with gel electrophoresis to see if PCR was successful.

  25. What happens when cells copy their own DNA? Polymerase Chain Reaction(PCR) This is the process mimicked by PCR. http://users.ugent.be/~avierstr/principles/pcr.html http://www.dnalc.org/ddnalc/resources/pcr.html

  26. Developed by Dr. Kary Mullis (1985) “Molecular Photocopying” • Allows scientists to make millions of copies of a selected stretch of DNA form within the total genomic DNA • Uses enzymatic amplification much like DNA replication, just at high-speed • Does not require splicing out the selected stretch, creating a vector, and cloning it in a bacterial or a yeast culture

  27. The Materials • DNA from a crime scene, from remains ,or from a suspected illegally poached animal (template DNA) • Pair of DNA primers that are approximately 20 nucleotides long and flank the target region to be amplified • Heat resistant DNA polymerase (Taq) from the thermophilic bacterium Thermus aquaticus • Magnesium (cofactor) • Four deoxyribonucleoside triphosphates (dNTPs) http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2002/Robinson/Isocitrate-main-page.html

  28. The Protocol • Denaturing Heat the mixture to 94oC to separate the template DNA into 2 strands (break hydrogen bonds). • Annealing Cool the mixture to 65oC so that the primers anneal (stick) to the template DNA. Primers anneal before the DNA template strands come back together, because they are short and present in excess. • Extending Heat the mixture to 72oC so that DNA polymerase can synthesize complementary strands from the primers. http://www.hhmi.org/biointeractive/dna/DNAi_PCR.html http://www.sumanasinc.com/webcontent/anisamples/molecularbiology/pcr.html

  29. The Product • Each cycle takes 2 minutes and doubles the number of template DNA molecules. • 25 cycles takes less than an hour and should produce 1,000,000 copies of template DNA molecules. • How many copies of template DNA would you have after 10 cycles of PCR? After 50 cycles? http://pathmicro.med.sc.edu/pcr/PCR_Reaction_graph2.htm

  30. The Machine • Once upon a time, scientists had to move reaction tubes among 3 water baths of different temperatures according to a strict timing routine. • Now, PCR is automated using a thermal cycler. • A computer controller raises and lowers the temperature of a heat block so that all tubes stay in one place, and the scientist can go out to lunch. http://www.labsupply.com.hk/Thermal%20Cycler%20001.htm

  31. RT-PCR • First, an RNA strand is reverse transcribed into its DNA complement (complementary DNA or cDNA) using the enzyme reverse transcriptase. • Then, the resulting cDNA is amplified using traditional PCR. http://archive.microbelibrary.org/microbelibrary/files/ccImages/Articleimages/Campbell/rt_pcr.html

  32. Lesson 6-7 Objectives: • Identify unknown white substances by creating a standard of color identification for known white substances and comparing these standards to the 5 unknowns. • Perform presumptive test or screening tests at the crime scene and at the lab to further analyze and confirm findings.

  33. Testing with Immunoassays • Immunoassays are used by CSI’s to analyze specimens for the presence of drugs. • CSI’s may use these tests at the crime scene or while collecting evidence. • Forensic Technicians may also use these procedures in a laboratory.

  34. Testing with Immunoassays • CSI’s use immunoassays in the field because they are fast, reliable, and consistently show a color to indicate the presence or absence of drugs, alcohol, or presence of bodily fluid at a crime scene. • In this investigation, a simple color indicator test will be used to demonstrate how colors indicate the presence of specific drugs.

  35. Procedure: • Take a wax pencil, and write on the lid of the cell culture container these solutions. Abbreviations for each solution can be written instead of the entire name. • Benedicts Solution • Fe(NO3)3 3.0 M • Hydrochloric acid 3.0 M • Iodine • Universal Indicator Color • Water Solubility • pH

  36. Place a small scoop, with the spatula, of one known white substance inside of each of these wells. • Choose one of the following: • Aspirin • Baking Soda Sodium Bicarbonate • Chalk • Contac • Cornstarch • Glucose • Sudafed • Salt • Flour • Tylenol

  37. Add 3 drops of each solution to the wells. Make sure you add the correct solution to each well. • 3 drops of Benedicts Solution • 3 drops of Fe(NO3)3 3.0 M • 3 drops of Hydrochloric acid 3.0 M • 3 drops of Iodine • 3 drops of Universal Indicator Color • 3 drops of Water • Place a pH strip into the well containing water after you have tested the substances solubility.

  38. Lesson 8-9 Objectives: Urine analysis thin layer chromatography • Evaluate evidence collected to identify what kinds of test or equipment are needed to analyze specific forms of evidence. • Analyze evidence, record data, and write conclusions

  39. Chromatography Basic principles • Require one static part (the stationary phase) and one moving part (the mobile phase). • Rely on one of the following: • Adsorption • Partition • Ion exchange • Molecular exclusion

  40. Chromatography Adsorption • Has a solid stationary phase and a liquid or gaseous mobile phase. • The different solutes, carried along by the solvent, will travel different distances through the solid. • Each solute has its own equilibrium. • The least soluble or best adsorbed ones travel slowly. • This results in separate bands containing different solutes. • The solvent that is put into a column is called the eluent, and the liquid that flows out of the end of the column is called the eluate.

  41. Thin layer chromatography (TLC) • The stationary phase is a thin layer of a solid like silica supported on an inert base such as glass, aluminum foil, or insoluble plastic. • The mixture is ‘spotted’ at the bottom of the TLC plate and allowed to dry. The plate is placed in a closed vessel containing solvent (the mobile phase) so the liquid level is below the spot.

  42. Thin layer chromatography (TLC) • TLC has advantages over paper chromatography: • Its results are more reproducible. • Separations are very efficient because of the much smaller particle size of the stationary phase. • The solvent travels up the plate by capillary action. • This process is performed in a closed container to make sure the environment directly around the experiment is saturated with solvent vapor, and evaporation from the plate is minimized before the experiment is complete.

  43. Thin layer chromatography (TLC) • The plate is removed when the solvent has reached the top of the plate, and the position of the solvent has been recorded before it is dried. • These values are needed to calculate the Rf value. • The solvent travels up the plate by capillary action. The liquid fills the spaces between the solid particles. This technique is usually done in a closed vessel to ensure that the atmosphere is saturated with solvent vapor and that evaporation from the plate is minimized before the run is complete.

  44. Thin Layer Chromatography (TLC) • Many spots are not visible without the plates being “developed.” This usually involves spraying the spots with a solution that is reversibly adsorbed or reacts in some way with the solutes. • Two examples of developing solutions are iodine in petroleum ether and ninhydrin (useful for identifying amino acids).

  45. Thin Layer Chromatography (TLC) • Iodine vapor is also used to develop plates in some cases. • Specially prepared plates can be used that fluoresce in ultraviolet light. Once dried, the plates are placed under an ultraviolet lamp. • Solute spots mask fluorescence on the surface of the plate as a dark spot is observed. Some compounds have their own fluorescence which can be used for identification.