Forensic Biology Final Exam

I decided to watch an analyst doing a serology-based exam on an unknown fluid. I have no clue what test is being conducted, but I know that they are looking for a visual change to indicate a positive reaction. From the tests we've discussed, what test are they NOT doing?

A mRNA-based assay

The main difference between precipitation and agglutination-based serological tests is…

Precipitation requires the antigen to be soluble while agglutination requires the antigen to be insoluble

I decided to watch an analyst doing a serology-based exam on an unknown fluid. I have no clue what test is being conducted, but I know that they are looking for a visual change to indicate a positive reaction. From the tests we've discussed, what test are they NOT doing?

A mRNA-based assay

The main difference between precipitation and agglutination-based serological tests is…

Precipitation requires the antigen to be soluble while agglutination requires the antigen to be insoluble

Serology tests that utilize DNA methylation can tell us:

• Whether an individual has a genetic disease

• From what species the DNA originated from

• From what fluid/tissue the DNA originated

What tests can and cannot be used for species identification?

Can be used to ID species (i.e. test is species specific)

• Crossed-Over Electrophoresis

• Ouchterlony Assay

• ELISA

Cannot be used to ID species (i.e. test is NOT species specific)

• Using Luminol (or Bluestar)

• Leucomalachite Green (LMG) Assay

• Takayama Assay

True or False: Immunochromatographic tests that use hemoglobin as a target to test for blood are prone to cross react with other biological fluids such as seminal and vaginal fluids. However, immunochromatographic tests that target the glycophorin A protein (GPA) to test for blood do not cross react with these fluids.

True

What statements about cross-reactivity are TRUE?

• On a molecular level cross-reactivity is the unwanted binding between an antibody and an antigen that has a similar conformation shape to our target antigen

• Cross-reactivity may occur between similar species (e.g. a test for human blood may cross-react with other non-human primate blood)

• Cross-reactivity may occur between biological fluids (e.g. a test for human blood may cross-react with human semen)

What statements about ELISA are TRUE?

• ELISA is a plate based assay that can detect substances such as peptides, proteins, antibodies, antigens, and hormones

• ELISA is not widely used in forensics anymore due to its time-consuming and cumbersome nature

• Immunochromatographic assays use a similar Ab-Ag-Ab (antibody-antigen-antibody) capture assay (i.e. Sandwich) technique that is used by ELISA

• The ELISA capture (i.e. Sandwich) assay is used over other ELISA techniques (e.g. Direct and indirect assay) due to its high sensitivity and specificity

In immunochromatographic assays (e.g. RSID kits) what portion/area of the antigen shown below binds to the antiglobulin found at the control zone?

Fc region

Which statement about antigens is NOT true?

All antigens induce an immune response.

The ouchterlony assay…

Is a double immunodiffusion assay in which the antibody and antigen are free to migrate

Primary vs. Secondary Binding Assays

Primary Binding Assays:

• ELISA

• More Sensitive

• Involves the initial binding between an epitope and an antibody

Secondary Binding Assays:

• Less sensitive

• Ouchterlony Assay

• Single Immunodiffusion

What could alter a presumptive assay that tests for the presence of blood resulting in false positives or negatives?

Oxidizing agents, plant peroxidase, and reductants

Complete the following sentence. Be as precise as possible: Chemically speaking the Phenolphthalein assay takes advantages of a(n) ___________, where _____________ serves as the catalyst to detect ___________.

redox reaction; heme; blood

What are colorimetric presumptive tests for blood?

• Phenolphthalein Assay

• Leucomalachite Green (LMG) Assay

• Benzidine Assay

• Kastle-Meyer Test

When analyzing blood what kind of an indicator does the analyst look for when performing microcrystal assays?

Crystalline formation

True or False: Aside from performing first aid (if necessary), the first critical step in crime-scene investigation is searching the scene for evidence.

False

Which type of evidence must be packaged separately from other evidence?

Biological stain evidence; DNA-containing evidence; Chemical evidence

True or False: The first DNA database was established in 1998 by the FBI (i.e. CODIS).

False

Police barricades, and the strategic positioning of guards, will prohibit access to the crime scene for:

All unauthorized personnel

The crime-scene notes must accurately record:

• The tasks assigned to each investigator

• Personnel arrivals and departures from the scene

• Descriptions of evidence present at the crime scene

True or False: A rough sketch does not need to depict the dimensions of the crime scene.

False

Messenger RNA (mRNA)...

Can be used for tissue identification

DNA collected from bone is most likely collected from what plentiful type of cells?

Osteoblasts

Explaining genetic copy number is sometimes tedious. Therefore, we often use a shorthand to quickly explain the genetic composition of a cell we are analyzing.Given this shorthand: 5N=115. Which of the following statements is NOT TRUE about the cell being analyzed?

The cell being examined is a somatic cell

Classify the following biological samples based on their nuclear DNA content and potential for DNA extraction. Match each sample to the appropriate category.

• Mature Red Blood Cells (RBC) - Low or no nuclear DNA content (difficult or impossible to extract)

• Blood Plasma - Low or no nuclear DNA content (difficult or impossible to extract)

• Leukocytes (White blood cells) - High nuclear DNA content (easily extractable)

• Dental Enamel - Low or no nuclear DNA content (difficult or impossible to extract)

• Dental Pulp - High nuclear DNA content (easily extractable)

• Hair Shaft - Low or no nuclear DNA content (difficult or impossible to extract)

• Hair Root (papillary tag) - High nuclear DNA content (easily extractable)

• Touch evidence - Low or no nuclear DNA content (difficult or impossible to extract)

• Osteoclasts - High nuclear DNA content (easily extractable)

• Osteoblasts - High nuclear DNA content (easily extractable)

True or False: Some DNA can be found outside of cells.

True

Which of the following immune responses are immunochromatographical assays based upon?

Acquired (adaptive) response

The velocity classification of a spatter pattern…

Can determine the general action that produced the pattern in regards to its speed of impact (e.g. handgun vs golf club)

Match blood spatter subjects to their definition.

• Angle of impact: The acute angle formed between the path of a blood drop and the surface which it contacts

• Back spatter: Blood directed back towards the source of the force that caused the spatter

• Cast-off: A bloodstain pattern that is created when blood is flung from a blood-bearing object in motion onto a surface

• Flow pattern: A bloodstain pattern formed by the movement of small or large amounts of blood due to gravity’s pull

• Impact spatter: A bloodstain pattern produced when an object makes forceful contact with a source of blood, projecting droplets of blood outward from the source

• Drip trail pattern: A pattern of bloodstains formed by the dripping of blood off a moving surface or person in a recognizable pathway separate from other patterns

• Forward spatter: Blood which travels away from the source in the same direction as the force which caused the spatter

• Satellite spatter: Small droplets of blood that are distributed around the perimeter of a drop or pool of blood that were produced as a result of the blood impacting the target surface

The ________ of a bloodstain pattern in a three-dimensional space illustrates the position of the victim or suspect when the stain-producing event took place.

Area of origin

Blood spatter that has been forced out through the trachea and out the mouth onto a surface is referred to as a(n)...

Expirated pattern

In general, as both the force and velocity of impact increase, what happens to the diameter of the resulting blood droplets?

Decreases

In what COUNTRY was the first crime lab established?

France

What are reasons why DNA polymorphisms replaced protein polmorphisms?

• DNA has a greater variability than protein polymorphisms

• DNA is found in most cells as opposed to proteins, which may be found in select fluids

• DNA can be amplified via PCR to yield more sample while proteins cannot be amplified

Which statement about note-taking is NOT true?

Note-taking is done after all crime scene processing has been completed

Order the following polymorphisms in order from oldest to most recently used (in regards to their use in human identification)

ABO Blood Group → Erythrocyte enzymes and serum proteins → SNP’s and STR’s

Forensic science is the application of science to:

Both criminal and civil laws

True or False: All evidence is collected at the crime scene

False

True or False: All evidence is collected at the crime scene

False

I decided to centrifuge a sample of whole blood so it separates out into several layers based on their densities. Match the following descriptors in the right hand column with the blood separated layer in the left hand column.

Erythrocyte Cellular Fraction (Portion):

• Contains heme

Buffy Coat Cellular Fraction (Portion):

• Contains the majority of DNA

• Contains platelets

Plasma (Portion):

• Majority is water

• Contains electrolytes

• Contains hormones

Match the following terms with the appropriate definition: Euchromatin, Allele, Short Arm (on a chromosome), Nucleosome.

Euchromatin: Loosely packaged DNA (more expressed)

Allele: Version of a gene or segment of DNA

Short arm (on a chromosome): 'p' arm

Nucleosome: A condensed packaging of DNA containing: DNA and histone proteins

A blood droplet falling onto a rough surface will have _____________ distortion around the edges while a droplet falling onto a smooth surface will have _____________ distortion around the edges. This difference is caused by changes to the droplet's ___________.

More, Less, Surface Tension

While conducting tests for the identification of sperm, you note that the suspect suffers from a condition known as aspermia. Which of the following tests of semen would likely result in a false negative due to this male's condition?

Microscopic Test

Sort the following tests for semen into either presumptive or confirmatory tests: Colorimetric tests (e.g. AP tests), Fluorescence tests (e.g. using ALS), RNA-based tests, and Immunochromatographic tests.

Presumptive:

• Colorimetric tests (e.g. AP tests)

• Fluorescence tests (e.g. using ALS)

Confirmatory:

• RNA-based tests

• Immunochromatographic tests

True or False: Microscopic tests for sperm require at least 5 spermatozoa to be present for the test to be considered positive.

False (only need 1)

Sort the following tests for saliva into either presumptive or confirmatory tests: Starch-Iodine Assay, Phadebas Assay, Microscopic Assay, and RSID-Saliva Kit (immunochromatographic).

Presumptive:

• Starch-Iodine Assay

• Phadebas Assay

• Microscopic Assay

Confirmatory:

• RSID-Saliva Kit (immunochromatographic)

Select the test/s that can positively identify vaginal epithelial cells from skin and buccal epithelial cells.

Dane’s Staining Method & RAMAN Spectroscopy

Christmas Tree Stain is most often used in the analysis of what biological fluid?

Semen

True or False: Immunochromatographic assays that target HSA or HPA can be used to detect the presence of saliva.

False

What is NOT a target used to identify urine?

Urobilin

Complete the following statement: Urobilin is created by the breakdown of ___.

Erythrocytes

Match the target to their respective fluid: Lactate Dehydrogenase, Creatine, Dermcidin, Acid Phosphatase, Salivary Amylase (HSA), and Pepsin.

• Lactate Dehydrogenase - Menstrual Blood

• Creatine - Urine

• Dermcidin - Sweat

• Acid Phosphatase - Semen

• Salivary Amylase (HSA) - Saliva

• Pepsin - Vomit

What are the primary targets for presumptive or confirmatory tests for saliva?

Buccal epithelial cells; RNA targets specific for saliva (e.g., HTN3); Amylase

Complete the following statement. ___ are the primary target for immunochromatographic tests targeting menstrual blood.

D-Dimers

What are the essential core building blocks of DNA?

Nitrogeneous base, phosphate group, pentose sugar

What are purines, what are pyrimidines.

Purines - A,G - 2 rings

Pyrimidines - C, U, T - 1 ring

I am trying to genetically modify a potato to make it darker to compete with Michigan State University and Lay's whiter potato chips (#PotatoChipDiversity). In doing so I try and create a "Franken-potato" that has more copies of DNA than it naturally has. Why would I do this? Well...why not?

Potatoes are naturally tetraploid and consist of 12 unique chromosomes.

I want to make a hexaploid potato (that also consists of 12 unique chromosomes). How would I write the ploidy of my darker pigmented 'franken-potato'?

Hint: Hex = 6; Tet = 4

Fill in the blanks to complete the ploidy shorthand (e.g., 2N=16) below.

6N = 72

How can you tell which end of a DNA strand is the 3’ end?

The 3’ end has a phosphate group

The genotype of an individual at a particular autosomal (i.e. non-sex chromosome) locus is labeled as follows: Aa. Which of the following statements about the individual at the aforementioned locus is TRUE?

The individual is heterozygous at the locus

What is the central dogma of biology?

DNA → RNA → Protein

In forensic applications Amelogenin is …

A protein whose primary focus is to help determine an individual's biological sex

I have a piece of double stranded DNA (dsDNA). How much cytosine content does the DNA have given there is 20% thymine content.

30%

Which of the following statements about the human genome is true?

There are many more copies of mtDNA in a cell than nuclear DNA

Classify the following genetic polymorphism: Individual 1: (GACA)(GACA)(GACA)(GACA) [4 copies of (GACA)]

Individual 2: (GACA)(GACA)(GACA)(GACA)(GACA)(GACA) [6 copies of (GACA)]

Length Polymorphism

Which of the following statements about restrictions enzymes is most true?

Restrictions enzymes cut at sites that are the same forward as they are backward

True or False: VNTR's are smaller than STR's.

False

VNTR's or _______ are MOST LIKELY to be found in _________ regions in the genome

Minisatellites; non-coding

I'm running RFLP on an individual. On the gel, I see a unique pattern of DNA bands that help identify the person. It's similar to creating a unique genetic fingerprint. These VNTR bands separate out on the gel based on what?

Length

What factors affect a DNA sample’s migration through an agarose gel?

• Gel composition (e.g. agar vs polyacrylamide)

• Gel concentration (e.g. 2% vs 5%)

• Sample size (i.e., DNA that is 1000bp vs 100bp)

What is one difference between the Single-Locus Probe Technique (SLP) and the Multilocus Probe Technique (MLP)?

SLP is better equipped to handle mixture samples than MLP

Which of the following best describes the difference between AFLP and RFLP?

AFLP is RFLP that has first undergone a round of PCR

What is a benefit of using SNPs over STRs to identify an individual? Select ALL that apply

• There are more SNPs available to pick from than STRs

• SNPs are not affected by degradation as much as STRs

• SNPs can be used in phenotype prediction (which can help ID an individual). STR's cannot be used for phenotype prediction

True or False: Single nucleotide polymorphisms (SNPs) are are always biallelic (i.e., having 2 alleles)

False

True or False: The first commercial kit designed to use SNPs as identifiable markers, PolyMarker, initially used a SNP, DQA1, that was highly variable in comparison to most other SNPs.

True

An indel can be…

An insertion or a deletion

Which of the following about Pm or Random Match Probability (RMP) is true?

RMP is the probability that the unknown sample matches the suspect

True or False: DNA Phenotyping analyses can be admitted into evidence in the US court system.

False

SNPs can be used to predict various phenotypes. Select ALL the phenotypes that can be predicted from SNPs.

• SNPs can be used to predict hair color

• SNPs can be used to predict height

• SNPs can be used to predict what a person’s face looks like

• SNPs can be used to predict geographical origin

Vocab matching! Match the term with its definition: Southern Blotting, Probe, Multiplexing, Restriction Fragment Length Polymorphism (RFLP) Assay.

Southern Blotting - Transferal of DNA from a gel to a membrane (or other solid matrix)

Probe - Anything that allows us to 'see' something that is ordinarily not visible or noticeable

Multiplexing - A reaction that involves running multiple samples in the SAME reaction

Restriction Fragment Length Polymorphism (RFLP) Assay - A mostly historical technique that serves as the foundation for DNA fingerprinting

Select ALL the goals of DNA extraction for use in forensic identification.

I.e., your only goal is to obtain a genetic profile from a piece of evidence for later comparison to a reference sample

Note: ONLY give me goals of DNA extraction

• To NOT extract RNA, proteins, salts, and other cellular 'gobbly-gook' that could complicate downstream analyses or serve as inhibitors

• To efficiently extract DNA from evidence (e.g. fluids, tissues, etc.)

Match the following components of a nucleotide with their general function.

Nitrogenous base - The component that encodes genetic information

(deoxy)Ribose Sugar - The component whose carbon elements are numbered (e.g., 3'-OH)

(tri)phosphate Group - The component that serves as the energy source for adding additional nucleotides to the nucleic acid

What is a primer dimer?

Two primers that self anneal to each other

True or False: We may perform PCR twice in the analysis of a genetic profile. Once to quantify (e.g. real time qPCR) and then later to amplify specific loci of interest for analysis (e.g. a form of end-point PCR). Given the fact above, evaluate the accuracy of the following statement:

Primers are always incorporated into the final PCR product (i.e. the resulting amplicon), in both real time qPCR AND in end-point PCR

True

Primer specificity and annealing temperature are very important when considering primer design. Which of the following about primer specificity is true?

Longer primers corresponds with higher annealing temperatures which corresponds to an increase in primer specificity (i.e. primer stringency)

What is or are method/s to remove (or at least control) the negative effects of a PCR inhibitor? Select ALL that apply.

• Perform an inhibitor separation prior to performing PCR (e.g. use a Centricon filter)

• Add a more robust polymerase

Select ALL statements that are true about DNA replication and PCR.

• Only DNA replication uses a topoisomerase to unwind/unkink downstream DNA

• The length of the amplicon (i.e., DNA product that’s the result of an amplification or replication event) is much shorter in PCR than in DNA replication

The following are all components that should be added to PCR EXCEPT…

All are components that should be added to PCR (MG+, Template DNA, dNTPs, inhibitors to PCR inhibitors e.g. Bovine serum albumin)

True or False: Intercalculating dyes such as SYBR Green, only bind to and fluoresce with ssDNA (i.e. single stranded DNA)

False

True or False: The Slot Blot technique can differentiate human DNA from non-human DNA

True

During which amplification phase do we measure PCR product formation (i.e. when we measure Ct) during quantitative real time PCR?

Exponential amplification

True or False: The Qubit is capable of measuring protein, DNA, and RNA concentrations simultaneously (i.e. in a single reaction)

False

True or False: In general, using the Nanodrop results in more accurate measures than using a Qubit.

False

After quantitating DNA an analyst may do all of the following EXCEPT....

Adjust the PCR amplification reaction in response to excessive DNA concentrations by increasing the amount of PCR cycles

What are goals of DNA quantitation?

• Determine the amount of total DNA

• Determine the amount of human specific DNA (Clarification Note: Granted some quantitation techniques cannot discern between total and human specific DNA. Technique aside, is this still a goal of quantification?)

• Determine the purity of DNA

Direct PCR methods are innovative in that they…

Are resistant against ultra low and ultra high amounts of DNA found within samples

True or False: In general, differential cell lysis is the most utilized method of separating a mixture sample that contains a perpetrator's sperm and victim's epithelial cells

True

Select ALL of the ways to reduce contamination.

• Use pipette filter tips when pipetting

• Apply bleach and alcohol to a workspace before performing an analysis

• Establish a Pre-PCR room as well as a Post-PCR room

Which of the following is a goal of DNA EXTRACTION?

Remove PCR inhibitors

What are components found in a lysis buffer?

• A detergent (e.g. SDS)

• Buffer (e.g. Tris)

• Chelating agents (e.g. EDTA)

There are many pros of using FTA for extraction. Which of the following statements about FTA cards is FALSE?

FTA paper is best used with non-liquid evidence