Forensic DNA Analysis

1. Forensic DNA Analysis DNA is the Genetic Material

2. Sources of Biological Evidence Saliva Blood Tissue Urine Semen Hair Feces Bone Teeth Skin Cells Sweat

3. Critical to Locate Evidence • It is important to have good crime scene analysis. • Evidence technicians, trained in DNA work, extract material from evidence. (BS in Science) • Crime Scene analysts identify evidence at the crime itself. (BS in Criminal Justice) • Forensic DNA analysts extract the DNA and perform the reactions and analyze the results. (MS in science)

4. What is Blood? • Slightly alkaline fluid made up of water, cells, enzymes, proteins, glucose, hormones, organic and inorganic substances • Circulates throughout the body • Supplied nutrients and oxygen to the body • Removes waste

5. Blood Cells • Cells mature and differentiate into several classes of cells • Red blood cells • White blood cells • Platelets

6. Red Blood Cells • Also known as Erythrocytes • Have no nucleus • Therefore note useful for DNA analysis • 6-8 um in size • ~45% total volume of blood • Most abundant cell in the blood

7. Red Blood Cells

8. White Blood Cells (WBC) • Also known as leukocytes • Produced in bone marrow • WBCs have a nucleus • Useful for DNA analysis • Vital source of defense against external organisms • White blood cells also clean up dead cells and tissue debris that would otherwise accumulate and lead to problems.

9. Red and White Blood Cells RBCs WBCs

10. Platelets • Irregularly-shaped, colorless bodies produced in the bone marrow • Their sticky surface lets them, along with other substances, form clots to stop bleeding. • Only active when damage occurs to the circulatory system walls.

11. Plasma • Liquid potion of blood • Composed of water, proteins, electrolytes • Blood cells and platelets are suspended in plasma • Regulates osmotic pressure • The transport medium for • Glucose, lipids, hormones, clotting factors, waste

12. Serum • Clear liquid that is left after blood coagulates • Plasma without the clotting factors

13. Analyzing Genetic Variation in Blood Forensically • Hemoglobin (RBC) • Peroxidase-like activity can cleave H2O2 • Blood Group Antigen (RBC) • ABO groups (on surface of RBC’s) • DNA (WBC) • Found in nucleus of WBCs • Proteins (Plasma) • Serum used in species testing

14. Blood Typing • All Blood groups are determined by the antigens on their red blood cells. • Antibody-B reacts with A-antigen and vice versa

15. Blood Typing • Rh factor • + Rh factor means your blood contains a protein also found in Rhesus monkeys (85% of people are Rh +) • Combined with the ABO system, the Rh factor can exclude suspects as well as include with some rarer blood types. • O+ 1 in 3 persons O- 1 in 15 persons • AB- 1 in 167 persons B- 1 in 67 persons

16. Presumptive Tests • Presumptive Tests are used to tell if a sample is blood. • Other presumptive tests can tell if a sample is human or primate blood. • This is important to avoid wasting time trying to purify DNA from red paint and meat juices. • Take a small rubbing from the stain, react it with chemicals and look for a color change.

17. Phenolphthalein Test

18. Phenolphthalein Test • Limitations • Sensitivity ~1/100,000 dilution • Lack of Specificity • Chemical oxidants (bleach), vegetable peroxidases cause reactions • Will not detect differences in animal or human blood • Stability • Relatively stable if the reagents are stored separately and refrigerated

19. Leucomalachite Green

20. Leucomalachite Green • Sensitivity ~1/1000 dilution • Lack of specificity • Chemical oxidants and vegetable peroxidases cause a reaction • Will not detect differences in animal or human blood • Stability- similar to Phenolphthalein

21. Hemastix • Reagent strips with TMB ends (Tetramethylbenzadine) • TMB changes from orange to green when it comes into contact with blood • Used for occult blood in stool • Somewhat quantitative • Very sensitive • TMB dangerous

22. Luminol

23. Luminol • How it works • The iron in hemoglobin acts as a catalyst to cause a reaction between the luminol and H2O2 • Luminol loses nitrogen and hydrogen and gains oxygen • This results in 3-aminopthalate which is energized and emits light

24. Luminol • Sensitivity ~10-8 (most sensitive presumptive) • Specificity • Bleach, metals, chemical oxidants, plant peroxidases light up too. • Will not differentiate animal and human blood • Very unstable ~4 hours for the mixture • Mostly used at crime scenes • Can dilute out blood

25. ABAcard Hematrace • Confirmatory test (shows that it is human or primate blood, since the other tests can be nonspecific) • Tests for human hemoglobin (Hb)

26. ABAcard Hematrace

27. Semen • Most common crime with DNA evidence is sexual assault • Sperm have DNA which can be well preserved if dried on cloth.

28. Semen Composition • Semen is a fluid of complex composition, produced by the male sex organs • There is a cellular component, spermatozoa, and a fluid component, seminal plasma

29. Seminal Plasma • Composed of salts, sugars, lipids, enzymes, nutrients, proteins, hormones, basic amines (spermine), P30, flavins • Enzymes- Acid Phosphatase • P30 ( a prostate specific protein used in prostate cancer tests) • Flavins • The components originate from several sources, including seminal vesicles and the prostate gland

30. Sperm Cells • Sperm are the male reproductive cells • Each consists of a head, tail and mid-piece • In humans, the head is a tiny disc, about 4.5 um long and 2.5 um wide • The tail is about 40 um long, and is rapidly lost in ejaculate

31. Is a sperm a sperm? • Human sperm vs. animal sperm • Dogs have similarly shaped sperm, but are about three times larger than human sperm • Other animals have differently shaped sperm • Situations?

32. Presumptive tests for Semen • Semen stains fluorescent under UV light • It is common practice to visually assess items of evidence under UV light to located possible semen stains • What fluoresces in semen? • The intensity of the fluorescence can be affected by the substrate, concentration of the stain, and other body fluids • Identified on crime scene or in lab by Evidence Technician • What else fluoresces? • Fibers, coffee, food, detergent, most organic stains

33. Presumptive test: Semen • Acid Phosphatase Test • Human semen contains high concentrations of acid phosphatase (AP), which can therefore be the basis of the screening test • While AP is detected in high concentrations in semen, it can also be detected in other body fluids • False positives • Vaginal acid phosphatase • Fecal material • Plant matter • Spermicides (orange) • Some feminine hygiene products

34. Confirmatory Tests: Semen • P30 identification • Found in semen • Microscopy • Identification of sperm

35. Prostate Specific Antigen (P30) • Antigen made in the prostate gland • Weighs 30kD • Liquefies semen and is instrumental in dissolving the cervical mucous cap for sperm entry • Early detection methods-electrophoretic double diffusion, Ouchterlony (precipitation band)

36. P30 Test

37. Sperm ID • Confirm the presence of semen by microscopically identifying sperm cells • Most common staining method is Kernechtrot picroindigocarmine stain- Alsp called Christmas Tree Stain • Prepared or commercially supplied

38. Human

40. Dog

41. Collection of Hair Specimen

42. Hair • Composed of cylindrical structures or shafts made up of tightly compacted cells that grow from follicles • Diameter ranges from 15-120 µm • Depends on type of hair and body region • Root material can be used for nuclear DNA testing • Shaft material can be used for some mitochondrial DNA testing too.

43. Hair Roots Pulled Forcibly Removed Shed

44. Tip of the Shaft Burned Cut Razored Split

45. Hair Basic Evaluation Steps • Determine if the sample is a hair • Determine if the hair is of human origin • Determine if the hair has root material-suitable for nuclear DNA analysis (Characteristic of a particular growth phase ) • If not suitable for nuclear DNA analysis, determine if the hair is sufficient in size for mtDNA analysis (2-3cm)

46. Hair • DNA analysis of hair is a destructive technique and results in the consumption of portions of the hair • Hair characteristics, such as color, length, shape, and texture should be noted in the case file for future reference prior to DNA analysis • Notes and digital images

47. Saliva Colorless fluid secreted by 3 glands in the mouth • Sublingual, submandibular, and parotid • Saliva from parotid glands contain amylases, enzymes, which aid in the digestion of carbohydrates • Saliva is composed of electrolytes, enzymes, mucus

48. Saliva Screening for saliva is based on detection of high levels of amylase in the sample • It is not a confirmatory test; amylase is found in other body fluids • Serum, urine, sweat, lip mucous, semen, feces, etc. • The concentration of amylase in saliva is variable among individual; if amylase is not detected in a sample it does not mean saliva is not present

49. Saliva • UV light can be used to aid in locating saliva stains • The intensity of the fluorescence can be affected by the substrate, concentration of the stain, and other body fluids • Saliva does not fluoresce as intensely as semen

50. Amylase • One of the earliest tests for amylase was the starch-iodine test • Iodine solutions cause starch to turn a deep blue color • Amylase is a starch hydrolyzing enzyme • The presence of amylase causes the disappearance of the blue color (due to hydrolysis of the starch) and can be used an indicator for the presence of amylase