Exam III Chapters 9, 11 and 20 Labs 9-12 Student Presentations

Chapter 9 Forensic Toxicology 1. What is toxicology? Toxicology- mix of Chemistry and Physiology that deals with drugs, poisons, and other toxic substances

2. What activities are considered part of forensic toxicology? Postmortem drug testing - Examples of this testing include overdoses and suspected poisoning Workplace drug testing - All Federal agencies require pre-employment drug testing - Some random testing of employees in specific occupations - 90% of largest U.S. corporations require drug-free urine as a condition of employment Evaluation of contraband material - Examples include seized drugs, methamphetamine or other clandestine drug labs

3. What is the Controlled Substances Act and how does it classify drugs? Federal Law established 5 schedules of classification of controlled substances based on – drug’s potential for abuse – potential to physical and psychological dependence – medical value

4. How do schedule I, II, III, IV and V drugs differ? Schedule I- Drugs with high potential for abuse and addiction, NO medical value e.g. heroin, LSD, ecstasy, marijuana Schedule II- Drugs with high potential for abuse and addiction, have some medical value with restrictions e.g. PCP, cocaine, amphetamines, most opiates, some barbiturates Schedule III- Drugs with less potential for abuse and addiction, currently acceptable for medical use e.g. some barbiturates, steroids Schedule IV- Drugs with low potential for abuse and addiction, currently acceptable for medical use e.g. tranquilizers like Valium, Xanax, Librium Schedule V- Drugs with low potential abuse, medical use, lowest potential dependency e.g. some opiates with non-narcotic ingredients

5. What is a poison? What is meant by the term “sufficient quantities”? poison- any substance that when taken in sufficient quantities causes a harmful or deadly reaction. (sufficient quantities- how much enters the body, over what period of time)

6. What is the difference between an intoxicant and a true poison? Intoxicant requires an ingestion of large quantities before it is lethal e.g. carbon monoxide, alcohol, heavy metals (mercury, lead, selenium) True Poison requires only a tiny amount e.g. cyanide

7. What does a forensic toxicologist do? A forensic toxicologist finds toxins and determines the likely effect on the individual who ingested or came in contact with it. Examples: -inebriation in an automobile accident or industrial accident -whether a person died from poison or from natural cause -whether drugs played a role in a perpetrator’s actions or in seizures or comas

8. Why are toxins described as “sneaky”? Effects of toxins do not cause VISIBLE changes in the body in living people or during an autopsy. Biotransformation can change one chemical into another within the body due to metabolism so the toxicologist may have to look for a different sign of the toxin.

9. What are the best places in the body for testing samples? What are the advantages or disadvantages of each place? Blood- most useful… tells what is going on at the time of death Urine- easily obtained… urine can’t be used to determine whether a drug was exerting any effect at time it was collected Stomach Contents- survivors are sampled by way of a gastric tube. Still no correlation between effects at time it was collected, but will determine if it was in the body. Liver- important in metabolism (destruction) of undesirables in the body. Many drugs concentrate in the liver, so there may be no sign in the blood, but the liver will reveal if a toxin is present.

Vitreous Humor- resists decay…may be the only fluid left in a decaying body, levels in this fluid lag behind the blood levels by 1 to 2 hours Hair- absorbs heavy metals and provides timeline of ingestion… can determine if a poisoning was quick (acute) or drawn out (chronic) - Arsenic will kill whether given all at once or slowly administered Insects- those that feed on the corpse will have elevated levels of certain drugs and may provide information if the drug was present at time of death

10. What are color tests? Know how the Marquis and Duquenois-Levine Tests are used to detect specific drugs. Color Tests- a reagent is added to the substance to be tested and a color change will represent the presence of the suspected chemical Marquis Test - turns purple in the presence of heroin, morphine, opium - turns orange-brown in the presence of amphetamines Duquenois-Levine - turns purple in the presence of marijuana

11. Know how immunoassays, gas chromatography, ultraviolet (UV) spectroscopy, and mass spectrometry (MS) are used. Immunoassays- antigen-antibody reaction…substance being sought is the antigen, testing reagent is the antibody Gas Chromatography- separates compounds according to size, shape, and chemical properties… will determine class of compounds but not exact makeup -can be used to separate a large mixed sample for further analysis Ultraviolet (UV) Spectroscopy- different compounds absorb and reflect light differently - can be used to indicate the concentration of a drug in a sample Mass Spectrometry (MS)- high energy bombards the sample and breaks it into tiny fragments. They are passed through a magnetic field and separated by molecular weight… - produces the same pattern so can be used for identification

12. How can toxins be administered and how does this affect where they are found in the body? Toxins will be most concentrated at the point of entry. Ingested Toxins- show up more in the stomach, intestines, and liver Inhaled Toxins- show up in the lungs Injected Toxins- linger in tissues around point of injection… muscle slows down the transfer to the bloodstream. Intravenously (IV)- will bypass the stomach and liver going directly into the bloodstream, and is therefore, quickly distributed… very little remains in the IV site - may find high concentrations in tissues but none in stomach or intestines

13. Why is the route of entry important to determine? Route of entry is EXTREMELY important in cases of injection where the person had no means or physical capabilities to inject … homicide is a stronger consideration.

14. Know the difference between normal, therapeutic, toxic and lethal concentrations. What is the LD50? Why are these categories not exact? Normal- expected in the general population under normal circumstances Therapeutic- the level your doctor wants in your bloodstream for medical reasons Toxic- a level that may cause harm- nausea, vomiting, change in heart rhythm, etc. Lethal- level at which the toxin consistently causes death -LD50 means the toxin at that level will cause 50% of people to die Wiggle room between categories- everyone reacts differently based on age, sex, body size, genetics, and health.

15. What are the responsibilities of the toxicologist? Know the chemical make-up, physiological actions, and by-products of the drug Understand how the drug is metabolized by the body and the potential of the metabolites Know how the chemicals will effect healthy people and people with various illnesses and addictions Recognize the symptoms and signs produced by the chemicals

16. Know the differences between familiar poisons like Cyanide- very lethal; damages internal workings of the cell; gives BRIGHT cherry red color to victim’s blood Strychnine- plant-based rat poison with extremely bitter taste; causes a LOT of pain and convulsions; death is by asphyxia; rigor mortis is quick because of the depletion of ATP during convulsions Mushrooms- easily confused with the edible type; causes severe damage to liver and extremely low blood sugar level Ethylene Glycol– antifreeze; causes deadly breakdown of oxalic acid reacting with calcium in the blood; crystallizes in the brain and kidneys Oxalic Acid– found in raw rhubarb; powerful irritant to gastrointestinal tract that may cause bleeding; low levels of calcium will be found in the blood Heavy Metals- e.g. arsenic, mercury, lead, antimony, bismuth, thallium - all behave slightly different but cause gastrointestinal injury and lead to nausea, vomiting, and diarrhea (bloody; damage kidneys, liver, brain, and nerves

Insulin- drops blood sugar level and robs brain of nutrients; can be caused by a tumor in the pancreas or injection Succinyl Choline- injectable drug that paralyzes all the muscles of the body; leaves behind little evidence of presence so ME must look for metabolites Corrosive Chemicals- strong alkali bases (lye) and acids that corrode and burn tissues of the digestive tract; cause bleeding, shock, and death

17. Know the differences between drugs considered to be depressants, stimulants, hallucinogens, narcotics and anabolic steroids. Depressants- make you sleepy and lethargic; considered “downers” e.g. alcohol, opiates, barbiturates Stimulants- increase alertness, lessen fatigue, suppress appetite; also associated with irritability, anxiousness, aggressive behavior, paranoia, fatigue, depression e.g. amphetamines, cocaine Hallucinogens- alter perception and mood; lead to delusional thinking and hallucinations. e.g. marijuana, cacti, mushrooms, LSD, PCP, Ecstasy, Rohyphonol Narcotics- pain reducers e.g. morphine, methadone, codeine, heroin Anabolic Steroids- increase testosterone; cause aggressive behavior and diminished sex drive; increase risk of liver cancer

Lab 9 Analysis of Drugs and Poisons 18. What is pH? What pH levels are considered acidic? What pH level is considered neutral? What pH levels are considered basic? pH- concentration of hydrogen ions (H + ) < 7 acidic 7 neutral > 7 basic, alkaline

19. Be able to analyze the results of known and unknown samples to identify an over-the- counter drug.

20. What is LSD and how can it be identified? What is a false positive? Lysergic acid diethylamide (LSD)- a hallucinogenic drug derived from ergot, a fungus that grows on rye and cereals, or prepared synthetically - presumptive identification with UV light - false positives- substances other than one tested that also give a positive result

21. What is marijuana and how can it be identified? Marijuana- dried portions of the Cannabis sativa plant whose active component is tetrahydrocannabinol - Crystals react with Hydrochloric acid (HCl) to produce carbon dioxide (CO 2 ) gas. - Duquenois Test is also used to identify marijuana based on characteristic color changes when mixed with the Duquenois reagent (acetaldehyde, vanillin and ethanol).

22. What are lead and mercury and how can they be identified? Lead- heavy metal that can be found in batteries, paint, gasoline and ceramic glazes - affects the function of the blood, liver, brain and kidneys. - damage to the kidneys can be detected in urine by large amounts of the amino acid alanine Mercury- heavy metal that can be found in batteries, thermometers, fungicides and industrial waste products; certain fish are also high in mercury. - accumulates in the brain and can lead to blindness, convulsions and even death - also affects the kidneys, leading to increased amounts of the amino acid glycine in urine

23. Be able to calculate the Rate of flow (Rf) in order to determine the identity of an amino acid and the type of heavy metal poisoning that occurred. Rf = distance traveled by amino acid distance traveled by solvent

24. What is DNA? What are the nitrogenous bases found in DNA? Be able to label the structure of DNA. Deoxyribonucleic Acid - polymer of nucleotides - nucleotide- monomer consisting of a sugar (deoxyribose), a phosphate group, and a nitrogenous base (Adenine, Guanine, Thymine and Cytosine) - Sugar-Phosphate backbones run antiparallel with the 5’ end of one strand across from the end of the other - 3 Hydrogen Bonds hold Guanine and Cytosine into a base pair - 2 Hydrogen Bonds hold Thymine and Adenine into a base pair

25. Who discovered the structure of DNA? James Watson and Francis Crick (using data from Rosalind Franklin)

26. What is a chromosome? What are autosomes? What are sex chromosomes? In its most condensed state, DNA and proteins form chromosomes

27. What is the difference between monozygotic and dizygotic twins? monozygotic (identical) twins- one zygote splits in two - identical DNA dizygotic (fraternal) twins- 2 different eggs are fertilized by 2 different sperm - DNA no more alike than two siblings

28. What is transcription? What is translation? transcription- a cell copies a gene’s DNA sequence into a complementary RNA molecule translation- the information in RNA is used to manufacture a protein by joining a specific sequence of amino acids into a polypeptide chain

29. What is RNA? What are the nitrogenous bases found in RNA? Ribonucleic Acid

30. What are proteins? proteins- one or more polypeptide chains of amino acids

31. Know how to use the genetic code to determine the amino acid sequence of a polypeptide when given the sequence of mRNA. e.g. AUG CAG GUC UCA UAG

32. What are mutations? mutations- changes in the DNA sequence e.g. sickle cell mutation

33. What are RFLPs? Restriction Fragment Length Polymorphisms (RFLPs)- sequences of nucleotides that are repeated in tandem different numbers of times in different individuals; also called minisatellites

34. How is DNA replicated? What enzymes are involved in the process? What is the difference between the leading and lagging strands? semiconservative replication- each new DNA double helix uses one strand from the original DNA molecule helicase- unwinds double helix and separates strands of DNA primase- adds an RNA primer to the start sites of replication DNA polymerase- adds new complementary nucleotides to the 3’ end - leading strand- replication proceeds continuously - lagging strand- replication proceeds discontinuously, producing Okazaki fragments, short pieces of DNA ligase- joins pieces of DNA together

35. What is PCR? What reagents are needed for PCR? What steps are involved in PCR? How does PCR change the amount of a specific DNA sequence? Polymerase Chain Reaction (PCR)- method for replicating DNA in vitro - template DNA - primers - nucleotides - Taq DNA polymerase

Step 1: Denaturation- high temperatures used to separate two strands of DNA Step 2: Annealing- primers hybridize, or bond, to complementary target DNA sequence Step 3: Synthesis- new nucleotides are added according to sequence of template DNA Each cycle, or round, of PCR doubles the target DNA sequence 2 n where n equals the number of cycles

36. What are STRs? Short Tandem Repeats (STRs)- sequences of 3- 7 nucleotides that are repeated in tandem different numbers of times in different individuals; also called microsatellites

37. What is capillary electrophoresis? A technique used to determine the size, and thus number of repeats, in a segment of DNA

38. What is CODIS? How many standard STRs are used by CODIS? Combined DNA Index System (CODIS)- computer software program developed by the FBI that maintains local, state, and national databases of DNA profiles from convicted offenders, unsolved crime scene evidence, arrestees, and profiles of missing people Uses 13 standard STRs

39. How can the sex of an individual be determined using DNA? Amel- gene coding for amelogenin, a product of tooth pulp, is 6 nucleotides shorter on the X chromosome than on the Y chromosome - males will have two bands or peaks - females will only have one band or peak Y- STRs- useful for mixed DNA samples

40. What is mtDNA and how is it used? mitochondrial DNA (mtDNA)- only inherited from mother so used to establish maternity Each cell contains multiple mitochondria and each mitochondrion contains multiple circular DNA chromosomes so mtDNA can be analyzed when nuclear DNA cannot as with some very old or decomposed remains.

41. How does DNA sequencing work? DNA is synthesized using both normal nucleotides and dideoxynucleotides tagged with different colors - When a dideoxynucleotide is added, DNA synthesis stops, creating DNA fragments of different lengths

Electrophoresis is used to separate molecules of different sizes - the shorter the DNA fragment, the faster it travels through a gel toward a positive charge Order of colored fragments of DNA is detected to reveal the sequence of nucleotides.

42. What are DNA chips or microarrays? a collection of short DNA fragments placed in defined spots on a slide After amplification, DNA is allowed to hybridize, or bind, to known fragments If DNA binds, it means that variant is present in the sample

43. What are SNPs? Single Nucleotide Polymorphisms (SNPs)- single nucleotide differences between individuals

44. How should biological evidence be collected and handled? Before the collection of biological evidence begins, it is important that it be photographed and recorded on sketches. Disposable gloves must be worn while handling the evidence. Biological evidence should not be packaged in plastic or airtight containers because the accumulation of residual moisture could contribute to the growth of DNA-destroying bacteria and fungi. Stained items should be packaged separately. All biological evidence should be refrigerated or stored in a cool location until delivery to the laboratory.

45. What are substrate controls? What are standard/reference DNA samples? substrate controls- obtained from an area adjacent to the stain standard/reference DNA- collected from a blood sample or a buccal swab

Lab 10 DNA Collection 46. What is a buccal swab? What types of cells are found on the inside of your cheeks? Where is DNA located in your cheek cells? buccal swab- collects epithelial cells from inside mouth (cheek) - DNA located in the nucleus (and the mitochondria)

47. What does a micropipette do? Why must pipette tips be changed between reagents? micropipette- transfers small (microliter) amounts of liquids Tips must be changed between reagents to prevent contamination!

48. What does a vortex do? What does a microcentrifuge do? vortex- vibrates to mix materials microcentrifuge- spins small (micro) tubes to separate materials

49. What does the term supernatant mean? supernatant- liquid on top after separation

Lab 11 PCR & DNA Fingerprinting 50. What reagents are needed for PCR and what does each do? DNA template- DNA that is copied, or amplified forward and reverse primers- needed to begin synthesis of both strands of DNA Taq DNA polymerase- enzyme from bacteria that synthesizes new DNA deoxynucleotide triphosphates (dNTPs)- building blocks for new DNA buffer- keeps pH stable during reaction distilled water

51. What is the purpose of the different temperatures in PCR? °C Denaturation- high temperatures used to separate two strands of DNA 60°C Annealing- primers hybridize, or bond, to complementary target DNA sequence 70°C Synthesis- new nucleotides are added according to sequence of template DNA

52. How many copies of the target DNA sequence will be generated from one copy of template DNA after n rounds of PCR? Each cycle, or round, of PCR doubles the target DNA sequence 2 n where n equals the number of cycles

53. Why is the amelogenin locus analyzed? Sex determination- product is 6 base pairs (bp) shorter on the X chromosome than the Y chromosome

54. What is CODIS? How many standard STRs are used by CODIS? Which standard STRs did we analyze? Combined DNA Index System (CODIS)- computer software program developed by the FBI that maintains local, state, and national databases of DNA profiles from convicted offenders, unsolved crime scene evidence, arrestees, and profiles of missing people Uses 13 standard STRs including von Willebrand factor type A (vWA) and D13S317

Lab 12 DNA Gel Electrophoresis 55. How is DNA separated by gel electrophoresis? charge- DNA is negatively charged and so travels toward the positive electrode size- smaller DNA fragments travel faster, and therefore, migrate farther through the gel

56. What is a DNA ladder, or marker? DNA ladder, or marker- contains standard fragments of DNA for size comparison

57. What is the purpose of using negative controls? negative controls- should give a negative result unless there is contamination

58. Know how to analyze DNA fingerprinting results to determine which suspect’s DNA matches DNA found at the scene of a crime.

Chapter 20 The Future 59. Why is there a need for trained and knowledgeable evidence collectors? Forensic science begins at the crime scene If evidence is not collected, or not collected properly, it will not be helpful for an investigation.

60. What overburdens crime labs? Disproportionate amount of drug and blood- alcohol evidence

61. How will undergraduate and graduate programs in forensic science affect the field? Better educational opportunities More opportunities for research

62. Why is the demand for DNA profiling increasing? Technology becoming faster and cheaper Laws to collect DNA profiles from arrestees Value in processing less serious crimes

63. How have computer databases helped investigators? Providing statistics for significance of evidence Generating suspects in investigations

64. Why must more time and effort be devoted to class evidence? Providing statistics for significance of evidence Several pieces of class evidence can be combined to strengthen case

65. What is essential for crime labs to develop? Standardized protocols

66. What important conclusion was made in the 2009 National Academy of Science report Strengthening Forensic Science in the United States: A Path Forward? Forensic science must be strongly rooted in science.