Forensic Biology Exam 2

What are the presumptive and confirmatory assays for semen?

Presumptive: Chemiluminescent/Fluorescent Assays, Colorimetric Assays

Confirmatory: Chromatographic & Electrophoretic, RNA-Based, MICROSCOPIC EXAMINATIONS

What are the targets for semen?

Acid phosphatase, prostate-specific antigen (PSA), seminal vesicle-specific antigen (SVSA)

Are visual exams (fluorescence) specific to semen?

No

Where is acid phosphatase found?

Seminal Plasma

Are Tests for AP specifc?

AP tests are sensitive, but NOT SPECIFIC

What do AP levels not affect?

Not affected by vasectomies

What is the AP presumptive test (fluorimetric)?

More sensitive and used to map stains

Colorimetric

What kind of reaction is AP?

Redox Reaction

What is a positive and false positive result of AP fluorimetric assays?

Positive = purple within 1 minute

False-Positive = purple after 1 minute

How many spermatozoa do you need to positively identify a sample in microscopy?

1 sample

How do you visualize semen samples under a microscope and which parts are stained?

Christmas Tree Stain

• Nuclei = red

• Neck and tail = green

• Acrosomal cap = pink

What is the easiest and fasted way to confirm spermatozoa?

Phase contrasting microscope

Where is the Prostate-Specific Antigen (PSA) produced and located?

Produced in the prostate

Located in the prostate fluids

Does a vasectomy have an impact on the detectability of PSA?

NO

Where can PSA ALSO be detected?

Urethral glands, perianal glands, sweat glands, and mammary glands

What are the most sensitive assays for PSA?

ELISA and Immunochromatographic assays

What is the advantage of Seminal Vesicle-Specific Antigen (SVSA) over PSA?

Concentration of SVSA is much higher than PSA (i.e. increased sensitivity)

Overall what confirmatory tests are used for the Identification of prostate-specific antigen (PSA)?

• Immunodiffusion

• Immunoelectrophoresis

• ELISA → most sensitive

• Immunochromatographic assays

Overall what confirmatory tests are used for the Identification of seminal vesicle-specific antigen (SVSA)?

• Immunochromatographic assays

• ELISA

What is HSA and what is it synthesized by?

Salivary amylases

Synthesized by salivary glands

What is HPA and where is it synthesized?

Pancreatic amylases

Synthesized by the pancreas and secreted into the duodenum

What can/cannot the measurement of enzymatic activity of total amylase distinguish?

Cannot distinguish HSA from HPA and nonhuman amylases

What can/cannot the direct detection of HSA proteins and RNA distinguish?

Can distinguish HSA from HPA

Human specific

What are the presumptive tests for saliva?

• Chemiluminescent/Fluorescent Assays

• Colorimetric Assays

• Microscopic Examination

What are the confirmatory tests for saliva?

• Chromatographic/Electrophoretic

• RNA-Based

For the Starch-Iodine test (saliva presumptive assay) how do the polymers interact in terms of color?

Amylose in starch reacts with Iodine to form a dark blue complex

Amylopectin reacts with Iodine to form reddish-purple color

In the presence of amylase starch is broken down, so NO color develops

Is the Starch-Iodine test specific to HSA?

No, can produce false positives

What is the central dogma of biology?

DNA → RNA → Protein

How do you view buccal epithelial cells (in saliva)?

Microscopy with aids of stains (e.g. iodine)

What are the layers of Human Vaginal Tissue and what do they contain?

Squamous mucosa (outermost layer): Made of stratified squamous epithelial tissue

Submucosa (under squamous mucosa): Contains connective tissue and capillaries

Muscularis (under the submucosa): Smooth muscle

What are the layers of the Squamous Mucosa and what do they contain?

Basal layer: large nuclei, anchored to basement membrane

Parabasal layer: cells begin to differentiate

Intermediate layer: flattened cells, compressed nuclei, glycogen in cytoplasm

Superficial layer: fully differentiated, small and dense nuclei, glycogen cytoplasm

What are the layers of vaginal tissue in this image?

What type of vaginal cells are normally swabbed?

Glycogenated epithelial cells

What is the importance of Dane’s staining method?

Can differentiate between skin, buccal, and vaginal epithelial cells based on color changes

What color do cells in Dane’s staining method stain?

Skin Cells: red and orange color

Buccal Cells: orange-pink with red nuclei

Vaginal Cells: bright orange with orange nuclei

What is RAMAN?

RAMAN is a spectrophotometric application that analyzes the energy given off (i.e., vibrational modes) of molecules when excited.

These energy spectra are highly unique to various molecules…

Including molecules found in bio fluids

What would be a positive result for the Lugol’s Iodine in Vaginal Secretions?

A positive test would be dark blue/red - opposite of saliva

Testing for intracellular glycogen not amylose/amylase

What are the presumptive tests for vaginal secretions?

ALS, Lugol’s Iodine, PAS Method, Dane’s Staining Method (can differentiate cells), and RAMAN

Essentially all assays are presumptive if you have to sort them

What is the main target for Menstrual Blood?

Clotting = D-Dimer

Can D-Dimers be human specific?

Yes, depending on how you make your antigen

What isoenzymes of Lactage Dehydrogenase (LDH) are predominant in menstrual blood?

LDH4 & LDH5

What are presumptive assays for urine?

• Visual Examination - Distinct color/odor

• ALS - urine stains emit fluorescent light

• Chemical analysis - detect phosphate, sulfate, urea, creatine, uric acid

  • NOT SPECIFIC

• Immunochromatographic assay

What are confirmatory test for urine?

• Identification of Tamm-Horsfall protein (THP)

• ELISE

• RSID-Urine

  • Presence of ALL 5 17-ketosteroid conjugates

  • Identified using liquid chromatography-mass spectrometry

What is the main target for urine?

Creatine - produced during normal muscle cell metabolism (not unique to urine)

What can detect creatine?

Uritrace device (Immunochromatographic assay)

What is urobilin?

Breaking down the heme from red blood cells leads to the formation of urobilin

How are urobilinoids detected?

Schlesinger test or Edelman test

• Cannot distinguish between human and other mammalian feces

What are some other assays used to identify fecal matter?

Macroscopic and microscopic examination - color and odor

Identification of fecal bacteria

What are some examinations for vomit?

Microscopic examination: identify recently ingested food (color may be informative red = fresh blood dark red = stomach bleeding)

Vomitus (Pepsin) Identification Assays: Load vomitus onto a gel that contains fibrin-blue (breakdown via pepsin releases the blue dye and causes a blue ring). NOT specific to humans

What are the two kinds of secretory sweat glands?

1. Eccrine (Activated by temperature) (higher up - superficial)

2. Apocrine (Activated by stress) (lower in the dermis - separate)

What type of sweat gland do we focus on in forensics?

Eccrine glands

What are some sweat identification assays?

Presumptive Assays (Detection of Lactic Acid): Scanning electron microscope + energy dispersion X-ray spectroscopy. NOT specific to sweat

Dermcidin - a potential biomarker for sweat. Specifically made in eccrine sweat glands. Antimicrobial peptide. Detected with: ELISA & mRNA assays

Who postulated the origin of genetics?

Gregor Mendel

What are Mendel’s laws of inheritance?

Law of Segregation: 2 members of a gene pair segregate ( = separate) from each other during sex cell formation, so ½ the cells contain the maternal gene, and the other ½ contain the paternal gene

The Law of Independent Assortment: Different segregating gene pairs behave independently due to recombination where genetic material is shuffled between generations

The Law of Dominance: For two alleles of a gene, the dominant allele is expressed and the recessive is masked

What was first thought to be the discrete unit of inheritance?

Genes

What was done to determine that DNA was in fact responsible for inherited phenotypes?

Griffith’s Transformation Experiment

Pathogenic strain = heat killed (proteins denature) = mouse lives

Conclusion: DNA is the transforming substance

What is DNA?

It is a nucleic acid (polymer of nucleotides)

• A pentose sugar (5 C’s)

• A nitrogenous base

• A phosphate group

What is the difference between Purine and Pyrimidine?

Purine = single ring

Pyrimidine = two rings

What is on the 5’ and 3’ end?

5’ end has the phosphate group

3’ end has the hydroxyl group

What is Chargaff’s Rule?

Double-stranded DNA molecule globally has percentage base pair equality:

%A = %T and %G = %C → BE ABLE TO CALCULATE

Can you perform Chargaff’s calculations on RNA?

No

What are genes?

The coding region of DNA in chromosomes that contains the information necessary for a cell to make proteins

What is a locus?

The chromosomal position or location of a gene or a DNA marker in a non coding region is commonly referred to as a locus (plural loci)

So if I asked you to find 3p22.1 where would you start looking?

Chromosome 3, P arm, band 2, subband 2, and subsubband 1

What is a homologous chromosome?

Pairs of chromosomes are described as homologous because they are the same size and contain the same genetic structure, a copy of each gene resided here (expect sex chromosomes - hemizygous/half the same)

What is a karyotype?

The process of pairing and ordering all the chromosomes of an organism, providing a genome-wide image of a person’s chromosomes

What is the difference between a sequence and a length polymorphism?

What is amelogenin and what does it help determine?

Amelogenin (the sex gene) is a protein that functions in the deposition of tooth enamel

• Used for sex determination

What is a SNP?

Single Nucleotide Polymorphisms

How long (bp wise) are SNP’s. How many alleles do they normally contain?

1 bp and 2 alleles

What are the 3 main ways that makes a genomic sequence diverse?

1. Variability/Complexity

2. Length

3. Number of Sites

Would a ‘fixed’, non-variable region be a good predictor of identity? Why?

If it is fixed and non-variable it is not changing therefore there is no diversity (hard to identify since there is no change, individual will have less fitness and will not reproduce)

What is the complexity of SNP’s and STR’s?

SNP’s: Normally biallelic, occasionally tri

STR’s: Dimeric (2) through hexameric (6); Tetrameric (4) (motif = repeat ex: AGAT)

Would a triallelic SNP be a better predictor than a biallelic SNP (assuming all allele frequencies are equal)?

Yes (one gives you more options)

What about dimeric vs. hexameric STR’s?

It doesn’t matter (it’s not the length it’s the number of times it gets repeated)

What are Indels?

Insertion-Deletion Polymorphisms = DNA segments of 1-100s nucleotides that are inserted/deleted

Easily Typed (like SNP’s) .May be Useful for Future Genetic Studies

E.g. human identity testing, phenotypic testing, etc

STR markers can be thought of as Multi-Allele Indels → Basically insertions/deletions of a tandem repeat unit

What do we want RMP to be?

Small

What is Pm?

Population Match Probability (better known as Random Match Probability, RMP)

Probability of an unrelated person randomly picked out of the general population will match the genotype from the evidence

What are some disadvantages of SNP's?

• PolyMarker relies on probes designed for specific SNP variations (more variations ~ more probes)

• Adding more probes causes the probes to bind to each other and not the designated area

• Complexity of SNPs << STR’s

• More SNPs needed to match informativeness of STRs

• Challenging mixture interpretations (due to biallelic signals)

What are some advantages of SNP’s?

• Very useful for degraded samples

• Higher level of multiplex capabilities over STRs

• No stutter = clear allele cells

• Phenotypic preditions

What are some current SNP applications?

• Human identification SNPs

• Acenstry informative SNPs

• Lineage informative SNPs

• Phenotypic informative SNPs

Will SNPs ever usurp STRs as a human identifying marker? Why or why not?

SNPs are very useful for additional sample information to be used mostly to supplement STR typing.

Standardization of SNP loci is still needed to be used for human identity testing applications.

Can SNPs be used to identify an individual?

SNP’s can’t compete with the identification potential of STRs. But, they can predict certain visible traits better than STRs

What are 3 factors that make a genomic feature identifiable?

genome size, number of genes, and chromosome number

What is RFLP?

Restriction Fragment Length Polymorphism

What are the 6 steps necessary to perform RFLP?

1. DNA Prep

2. Restriction Enzyme Digestion

3. Gel Electrophoresis/Separation

4. DNA Fragment Transfer

5. Hybridization with Probes

6. Detection

What is a restriction enzyme and where do they cut?

An endonuclease that cuts on the inside

What affects resolution?

Mobility of the sample is determined by:

• Sample itself (size of fragments/polarity)

• Medium (composition & concentration)

What is a probe?

An entity that can ascertain if what you are looking for is actually there (fluorescent, colorimetric, radioactive)

What does the single-locus probe technique (SLP) do?

Recognize a specific region of the genomic DNA at a VNTR locus

Creates a simple pattern → a DNA profile

• One band: homozygous

• Two bands: heterozygous

What is it called when one probe can hybridize to multiple VNTRs?

DNA Fingerprinting

What is MLP?

Multilocus Probe Techniques

• Detects multiple VNTR loci simultaneously (typically G-C sequence)

• DNA Fingerprinting

  • Used in immigrant parentage cases

  • Not useful in mixed samples

    • Impossible to resolve

What are two methods to detect a RFLP probe?

1. Radioisotype labeling (x-ray film)

2. Enzyme-Conjugated probe (chemiluminescence)

What factors affect RFLP results?

• DNA Degradation (longer bp = more susceptible to degradation)

• Restriction Digestion-Related Artifacts

• Electrophoresis and Blotting Artifacts

• Cutting at the wrong spot (partial restriction digestion & star activity)

  • Partial = not cleaved enough

  • Star = too much cleaving

• Point Mutations (band is longer than allele, endonuclease will not cut)

• Bands running off gel

What is AFLP?

Amplification Fragment Length Polymorphisms

• Small enough to undergo PCR

• 16 bp in each repeat unit (14-42 repeat units)

• Fragments separated with polyacrylamide gel electrophoresis and detected with silver stain

What are some categories of STR Markers?

Simple repeats, simple repeats with non-concensus alleles, compound repeats, and complex repeats

What are simple repeats?

Contain units of identical length and sequence

Ex: (GATA)(GATA)(GATA)

13 CODIS Loci → TPOX, CSF1PO, D5S818, D13S317, and DI6S549

What are Simple repeats with non-consensus alleles?

Ex: (GATA)(GAT-)(GATA)

13 CODIS Loci → TH01, D18S51, D7S820

What are compound repeats?

Comprise two or more adjacent simple repeats

Ex: (GATA)(GATA)(GACA)

13 CODIS Loci → VWA, FGA, D3S1358, D8S1179

What are complex repeats?

Contain several repeat blocks of variable unit length

Ex: (GATA)(GACA)(CA)(CATA)

13 CODIS Loci → D21S11