Forensic DNA Analysis DNA is the Genetic Material

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

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)

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

Blood Cells Cells mature and differentiate into several classes of cells –Red blood cells –White blood cells –Platelets

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

Red Blood Cells

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.

Red and White Blood Cells RBCs WBCs

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.

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

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

Analyzing Genetic Variation in Blood Forensically Hemoglobin (RBC) Peroxidase-like activity can cleave H 2 O 2 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

Blood Typing All Blood groups are determined by the antigens on their red blood cells. Antibody-B reacts with A-antigen and vice versa Blood Type: Antigens on RBCs Antibodies in serum AAAnti-B BBAnti-A AB Neither Anti-A or Anti-B O Neither A nor B Both Anti-A and Anti-B

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

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.

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

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

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

Luminol

How it works –The iron in hemoglobin acts as a catalyst to cause a reaction between the luminol and H 2 O 2 –Luminol loses nitrogen and hydrogen and gains oxygen –This results in 3-aminopthalate which is energized and emits light

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

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

ABAcard Hematrace

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

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

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

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

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?

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

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

Confirmatory Tests: Semen –P30 identification Found in semen –Microscopy Identification of sperm

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)

P30 Test

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

Human

Dog

Collection of Hair Specimen

Hair Composed of cylindrical structures or shafts made up of tightly compacted cells that grow from follicles Diameter ranges from 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.

Hair Roots Pulled Forcibly Removed Shed

Tip of the Shaft Burned Cut Razored Split

Hair Basic Evaluation Steps 1.Determine if the sample is a hair 2.Determine if the hair is of human origin 3.Determine if the hair has root material- suitable for nuclear DNA analysis (Characteristic of a particular growth phase ) 4.If not suitable for nuclear DNA analysis, determine if the hair is sufficient in size for mtDNA analysis (2-3cm)

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

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

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

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

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

Phadebas ® Test Positive test Negative test

Vaginal Secretions Vaginal secretions are a complex mixture of cells and secretions There is no confirmatory test to identify vaginal secretions Several screening tests based on microscopy have been proposed.

Vaginal Secretions Vaginal epithelial cells are large, and many contain glycogen (a polysaccharide) which can be demonstrated by staining with iodine in the form of a solution or exposing to iodine vapor. The presence of glycogenated cells is variable depending on the stage of the woman’s cycle Glycogenated cells can be found in other body secretions (i.e. oral, anal)

Fecal Material Feces are food residues passed after completion of travel through the digestive system Has a characteristic odor mainly due to skatole, an organic compound that occurs naturally in feces

Fecal Material Microscopy Microscopy has been used to identify fecal material –Looking for undigested residues of food material Chemical Tests Detection of urobilinogen, a bile pigment excreted in feces, which may be detected using its fluorescent reaction to Edelman’s reagent

Urine No confirmatory tests for the presence of urine Urine stains fluorescent under ultraviolet light –This can be useful for locating stains prior to chemical testing Has a characteristic odor Contains a large amount of urea, a chemical byproduct of normal metabolic processes in the body –Identification of high levels of urea can serve as a screening test for urine in fluids or stains –Perspiration can give reactions similar to urine

Urine Contains creatinine, which is a breakdown product of creatine (an important part of muscle) Over time, the creatine molecule gradually degrades to creatinine Creatinine is a waste product that is excreted from the body entirely by the kidneys –Identification of creatinine can serve as a screening test for urine in fluids or stains

Creatinine Jaffe Test One of the oldest tests for the detection of creatinine-1886 Creatinine forms a red compound with picric acid (Jaffe test)

Teeth and Bone DNA can be extracted from nucleated cells preserved inside teeth. DNA can be preserved in bone marrow, especially if bones are dry. Teeth are drilled in the lab and the material is extracted and DNA is extracted. Bone is drilled out and DNA is extracted Teeth and bones are best tissue for DNA analysis from skeletal remains.

Touch Evidence Epithelial cells that have been transferred from the person to the evidence via “touching” These samples tend to have low amounts of DNA Would expect body fluid stains to have more DNA than touch evidence Body fluid comparison: –Undiluted semen (with sperm) stain > blood stain –Both blood and semen > saliva stain –Body fluids > wear area stain (armpits, collar of a shirt)

Case Processing Includes: Location and Collection of Evidence Collection Techniques Preservation of Evidence Packaging and Storage Documentation-Chain of Custody Casework Analysis

Location and Collection of Evidence Evidence: Physical Evidence - any tangible object that can connect an offender to a crime scene, an offender to a victim, a victim to a crime scene etc. Biological Evidence is physical evidence - but is not always visible to the naked eye

Sources of Biological Evidence Blood Semen Saliva Urine/Feces Hair Teeth/Bone Tissue Cells

Reference Samples Blood –Blood transfusions Buccal Swabs/Saliva Clothing –Last resort secondary standard Other Secondary Standards –Toothbrush, hairbrush, glasses, etc. Other standards –All persons who had access to a crime scene should be documented –May be necessary to collect samples from these individuals

Actions to Avoid Touching any areas/items where DNA may exist Touching your face, nose, hair, and mouth when collecting and packaging evidence Sneezing, coughing, excessive talking near evidence –Gloves and or masks should be changed if contaminated

Protect the Evidence Preserve evidence with proper packaging Proper storage Use appropriate labels BIOHAZARD LABELS Case#, Initials, Descriptive label, date packaged Sign across your seal!!

Evidence Handling Never collect evidence without documenting the location, conditions, etc first Sketches, photographs, detailed notes The fewer people who handle evidence, the better Decreases chance of contamination Assists in court admissibility hearing

Chain of Custody A record of individuals who have had PHYSICAL possession of the evidence Critical in maintaining the integrity of the evidence If DNA analysis results in a foreign DNA type, it may be necessary to identify persons who handled the evidence

Chain of Custody Components Identifiers that describe the evidence at the time it was found –Location –Position –Date/Time of Collection Packaging/Sealing information

Multi-Section Cases Many items need to go through other sections of the lab Common requests: –Latent Prints –Trace / Microanalysis –Firearms Important to decide based on case, which evidence has priority