Forensic Biology - Serology and Extraction

What percentage of genetic code is shared among humans?

99.6%

Importance of Human Genetic Variation

• helps to understand genetics and evolution beyond our species

• Understanding of genomic basis of disease, pharmacogenetics, and gene therapies

Identification looks at markers that vary between individuals which are:

• VNTRS

• STRS

• SNIPS

VNTRS (Variable Number Tandem Repeats)

• Minisatellites inherited codominantly (from each parent)

• Repeated stretches of a DNA sequences (6-100 bp)

Sir Alec Jefferys

• invented DNA fingerprinting in 1985 using VNTRs

• First case of Forensic significance: Andrew Sarbah Immigration Case 1985 (denied reentry as a British citizen and used fingerprints to confirm identity)

First Case of DNA Profiling

• 1983 and 1986 two women were raped and murdered

• In 1986 semen stain matched both cases and exonerated Richard Buckland as suspect

• Mass screening of 5000 men were found; Colin Pitchfork paid colleague to submit for him but was found guilty in 1987

First Identification using STRs:

• In 1991 Jefferys, Gray, and Hagelberg extracted DNA from a femur from a 15 yr old girl who was killed 8 years earlier

• Compared DNA to parents for a positive ID

STRs are considered

The gold standard for DNA profiling

DNA Profiling

Distinguishes individuals from the same species based on their DNA

DNA Fingerprinting

Used to describe the original DNA profiling method using VNTRs

Presumptive Tests

• establish the possibility that a specific bodily fluid is present

• Not conclusive

Presumptive Test Pros:

• narrows possibilities

• Used on larger areas

• Locate evidence not visible to the naked eye

Presumptive Test Cons:

• Risk of false positives

• Can be overly sensitive

Confirmatory Tests

• Conclusively identifies biological material

Confirmatory Test Pros and Cons:

• Pros: smaller risk of false positives and conclusive test

• Cons: may be more expensive, longer timeframe, and addition equipment may be necessary

Why do we use Serology?

• Link suspects and victims to crime scene and to each other

• ID weapons and corroborate case circumstances

• Establishment of the Crime Scene: human or nonhuman blood present

Alternate Light Source (ALS)

Assist in screening evidence via fluorescing stains allowing for visible detection, otherwise not visible with the unaided eye

Kastle Meyer Test (KM)

• Pink color change with the presence of hemoglobin

• Presumptive for blood

• chance of false positives

• no distinction from animal and human blood

Luminol and Bluestar

• spray that detects iron in hemoglobin

• presumptive for blood

• chance of false positives

• harder to obtain a DNA profile

Leuco-Crystal Violet (LCV)

• uses hydrogen peroxide to detect hemoglobin

• permanent blue/purple stain

• presumptive for blood

RSID Blood

• confirmatory for blood

• detects the presence of human glycophorin A

• use of colloidal gold conjugated mouse antibodies

ABAcard HemaTrace

• confirmatory for blood

• targets human hemoglobin

• cross reactivity with higher primates

Acid Phosphatase Test (AP)

• turns purple in detects alpha-naphthyl and phospate

• presumptive for semen

• false positives with older stains and other body fluids with AP

PSA or P30

• Presumptive for semen

• detects P30 in seminal fluid

• cross reactivities and chance of false positives

Microscopy

• confirmatory for sperm

• Christmas Tree stain

• other sperm species will stain but morphology will be different

Sperm Hy-Liter

• specific to human sperm

• stains only sperm heads (target for DNA)

RSID - Semen

• confirmatory for semen

• detects the presence of seminogelin

• unique to human semen

• Potential for High Dose Hook Effect (result in a weak positive or false negative)

Y-Screening (instead of Serology)

• approach emerged to address the backlog of sexual assault kits

• Leverages qPCR approaches

Phadebas Test

• confirmatory for alpha amylase

• presumptive for saliva

• Press test

• chance for false positives

RSID Saliva

• confirmatory for human alpha amylase

• presumptive for saliva

Sample Type: Hair

• made of keratin

• Anagen Phase (growing phase; best for nDNA samples)

• Catagen Phase (follicle becomes inactive)

• Telogen Phase (growth cycle ends; mtDNA)

Sample Type: Bone

• often a last resort sample

• spongey (degrades quickly) and compact bone (degrades slowly)

Locards Principle

“every contact leaves a trace”

TPPR Factors

Transfer, Persistance, Prevalence, Recovery

Common Steps for DNA Extraction

1. Disrupt Cells/Tissues

2. Inactivate DNAases and RNAases

3. Denature proteins

4. Separate Nucleic Acids

5. Maximize Quality and Quantity of Recovered DNA

Common PCR Inhibitors

• Heme

• Melanin

• Calcium ions

• Humic acid

Purpose of a Reagent Blank (RB)

Guards/detects contamination within the extraction workflow

EDTA

• Chelating agent

• deactivates DNAases by binding to calcium and magnesium

Proteinase K

• protease enzyme

• breaks down proteins

• deactivates DNAases

SDS (Detergent)

• lyses open the cell membrane

DTT

• breaks disulfide bonds in proteins

• semen and hair

Organic Extraction - Phenol Chloroform

• mixed with cell lysate and centrifuge to create an aqueous phase and a organic phase

• aqueous phase moved to new tube and phenol:chloroform is added

• aqueous layer is pulled out again for ethanol precipitation

• DNA becomes hydrophobic - becomes a pellet

Solid Phase Extraction - Silica Based

• DNA is dehydrated to maximize negative charge (with a chaotropic salt)

• silica binds and releases DNA dependent on salt concentration

• DNA selectively binds to solid material and impurities washed away

• purified DNA is released from the silica

Silica-Based Column

• allows positive ions to form a salt bridge between the silica and negative DNA backbone in high salt concentration

Bead Based Method

• Magnetic Beads (silica based)

• fast

• no organic extractions or precipitates

• amenable to automation

Blood Extraction (simple)

• removal of heme (PCR inhibitor)

• liquid and dry samples require little prep

Saliva Extraction (simple)

• buccal swabs

• DNA from epithelial cells

Semen Extraction (Moderate)

• DTT necessary

• protamines must break down in sperm to access DNA

Differential Extraction

• used when there is a mixture of semen and epithelial cells

• normal lysis with ProK and SDS (must occur first)

  • nuclear membrane of sperm remains unlysed

• remove epithelial cell supernatant and use DTT to lyse sperm

Follicle

• below the skin

• connected to blood vessels that supply nutrients to the papilla

• nerve cells wind around the follicle

Hair Shaft: Cuticle

scaly outer layer made of keratin

Hair Shaft: Cortex

primarily made of keratin and contains a middle layer of melanin

Hair Shaft: Medulla

inner layer that can also contain melanin

Anagen Phase Hair

roots contain active germ and stem cells with associated nuclear DNA

Follicular Tag

blood and tissue attached to the follicle which may be seen when hair is forcibly removed

Challenges of Hair

• very fragmented nDNA

• keratinization causes loss of nDNA in the cortex

• hair at the crime scene is often in the Telogen phase

Challenges of Bone

• hard tissue with high calcium content

• must decalcify to get DNA

• dependent on age and condition of bone

• smallest DNA yield of samples

Decalcification

use of an extraction buffer with a high concentration of EDTA to bind to calcium and ProK and SDS

Sample Type: Teeth

• pulp cavity and root (cementum) are rich of DNA

• similar to bone extraction but must use methods to obtain tissue from the pulp cavity or root for old samples

• can survive most postmortem events

Crude DNA Extraction - Chelex

• Cells are boiled in 5% Chelex 100 resin

• chelex protects DNA from damage while denaturing proteins/cells

• centrifuge to separate chelex from the DNA

• ensure all chelex is removed as it is a PCR Inhibitor (binds to Magnesium and prevents replication)

FTA Paper

Cellulose based filter paper which contains a chemical mixture that lyses cells, denatures proteins, protects against DNA degredation, and inhibits bacterial growth

Direct PCR

• DNA profiling without extraction

• only used on reference samples not case work samples