Bio Fluids & DNA Flashcards
The identification of blood typically employs…
presumptive tests & a confirmatory test
What supports the identification of blood?
Visual observation
Positive chemical presumptive tests
Confirmatory test
Catalytic tests employ…
the chemical oxidation of a chromogenic substance
Two most commonly used presumptive tests today
Kastle-Meyer & Hemastix
Phenolphthalein
Produces a bright pink color when used in testing suspected blood
Luminol
Produces light when reacting with blood
Chromogenic substances produce what when reacting with a material of interest?
A colored substance
The starch-iodine test is __ for saliva.
Presumptive
SAP can be found….
In semen, breast milk, saliva, and other fluids
A presumptive test for blood can result in a false positive from…
Chemical oxidants
Materials of animal origin
Plant material
Vegetable peroxidases
What characteristic does an enzyme need to be useful for PCR?
Withstand extreme heat
Who do you inherit your mitochondrial DNA from?
Your mother
A heterozygous person has how many peaks a particular locus?
Two
Presumptive Test
Test which indicates the presence of a compound of interest
-High sensitivity, moderate selectivity
-Material should be examined further
Confirmatory Test
Test or tests which positively identify the material
-Moderate sensitivity, high selectivity
The 5 test results
False positive
False negative
True positive
True negative
Inconclusive
Presumptive testing method steps
- Sample the stain (swab or filter)
- Apply the reagents
- Document results
Kastle-Meyer/Phenolphthalein Test
-Use on visible stains
-Reagent is phenolphthalein in an alkaline solution
-Peroxide will react with the iron in hemoglobin (if blood truly is present)
Leucomalachite Green
-Use in visible stains
-AKA McPhail’s reagent
-Add hydrogen peroxide to reagent + hemoglobin
Test Sticks
-Use on visible stains
-Uses tetramethylbenzidine as the reactive agent
-Touch the lad to a moistened stain; produces blue-green color with a positive result
Leucocrystal Violet
-Use on low visibility stains for enhancement
-Reacts with hemoglobin to turn purple/black
Luminous Tests
-Use for detection
-Luminol as reactant, makes blood glow (glow is very weak)
-Bluestar as another reactant, produces stronger glow
-Hemascein as reactant, makes blood fluoresce (weaker glow, requires filter)
Which oxidizers most commonly cause false positives?
-Bleach
-Copper & Iron
-Plant fluids
-Air (ozone)
Teichman & Takayama Tests
-Form hemochromogen crystals by reacting to iron in blood
Immunoassay Test
-ABAcard Hematrace
-Hexagon OBT1
-Like a “covid test” but for blood
Karl Landsteiner
-Wanted to figure out why some blood transfusions didn’t work
-Discovered/named main blood types
What are immunoassays used for?
-Drugs
-Hormones (Pregnancy tests)
-Cholesterol levels
-Fluid Identification
Ouchterlony precipitin test
-Different species’ antibodies and questioned blood
-Antibodies and antigens diffuse through gel, leaving a visible line
OBT1 & ABA Hematrace immunoassays
Confirm the presence of blood/tell if it’s human or not
Acid Phosphatase (AP)
Secreted in large quantities by the prostate and other organs
-Brentamine Fast Blue B is used to identify it (effective on men with low/no spent counts)
Time to detect sperm heads?
V: 7 days
A: 2-4 days
Mouth: 24 hours
Time to detect tailed sperm?
V: <26 hours
Everything else: <5 hours
Being a serologist requires
At least a bachelor’s degree of science
Serology is a dying field because
DNA analysts typically perform serological duties themselves
Fibrin
Protein that is formed after injury to form blood clots
The solid part of blood is made up of
Red and white blood cells, and platelets
Main difference between plasma and serum?
Serum has no clotting factors, plasma does
Luminol characteristics
-Lasts for about 30 seconds
-Can be performed multiple times
-Numerous false positives are common
Bluestar Forensic characteristics
-Lasts longer
-Environment does not have to be completely dark
-Common for false positives, but not as often as luminol
Fluorescein characteristics
-Requires an ALS to view properly
-Can be used in a heavily lit room
Electrophoresis Method
Electrically charged gel used to determine species origin of an unknown sample
There is no test kit for _____
Vaginal fluids
Types of presumptive tests for urine
ALS
Jaffe Test
SEM-EDX
Touch DNA
DNA that is left behind by an individual when they come into contact with an item
system of linked databases that start at the local level and move through the state level to the national level
CODIS
Homologous:
pair of chromosomes
Locus/Loci:
Location of a sequence on a chromosome
Alleles:
different possible sequences at a locus
Homozygous:
Two identical alleles at one locus
Heterozygous:
Two different alleles at one locus
DNA is found in any….
nucleated cell
Examples of nucleated cells
White blood cells
Epithelial cells
Bone Marrow
Hair roots
Tooth pulp
Spermatozoa
How might we collect DNA samples?
Swabbing:
Stain
Orifice
Tool
Buccal swab;
Cut sample:
Clothing
Bedding
We must keep samples ____
Dry & Cold
What do we do during the extraction stage?
Break down proteins
Remove cellular debris
Remove clean DNA
No two people have ____ genomes
identical
Variation in ____ is stable
non-coding areas
Sir Alec Jeffreys
geneticist who figured out how to target areas of the genome with great variability and individualize the source of biological material
More bands=
More power
Gel Electrophoresis requires
large amounts of high quality DNA
STR Process
Mix labeled primers with sample
Amplify via PCR
Use electrophoresis to separate the fragments
Compare the fragment sizes to a ladder of known alleles for each locus
Report the fragments as a profile
Compare the profile to a sample or database
Strengths of STR
Looks at specific loci
1 bp resolution
Fast
Can be multiplexed
Can be databased
Done by a robot rather than a human
Most of the tests used at crime scenes are presumptive tests, which have a certain likelihood of false positives. Why is this OK?
Even if we accidentally collect non-blood material, confirmatory tests conducted at the lab will collect it.
What are chemical markers?
Antigens
Individual cells can have ___ antigens
Many
What is the relationship between antigens and antibodies?
Antibodies recognize and attach to highly specific types of antigens
We are using the OBT1 to determine if a blood sample is human or not. The control area of the test does not change color.
Which of the following statements about this test is true?
The mobile antibodies did not bind to the immobile antibodies in the control zone.
Which is the appropriate choice for initial survey for and detection of semen stains?
Visual detection with ALS
On their own, either the acid phosphatase or prostate-specific antigen test would be considered a ___ test for semen
presumptive
Which tests can be used to reliably identify saliva?
Phadebas
RSID Immunoassay
Starch Iodine Test
The two best ways to detect the presence of urine is the use of the ____ or the presence of ____
ALS; odor
Bleach
Oxidizes heme-reactive blood tests, causing a false positive
Hematrace
Immunoassay that reacts to proteins on the outside of the red blood cells
Takayama test
Confirmatory test which produces hemochromogen crystals
Hemastix
A self contained test pad on a plastic strip, used with visible stains
McPhail’s Reagent
Reacts with the iron in the hemoglobin to turn a suspected visible or faint blood stain green
What does the PCR process do?
Makes a BUNCH of copies of something
Microvariant
An allele with an incomplete repeat
Capillary Electrophoresis
Thin tube packed with silica beads that detects individual base pairs in fragments
Multiplexing
Sorts all loci at once
Loci with similar length alleles are given a unique color
If we have an identification….
All the known’s allele’s are present in the unknown
There are no extra alleles in the unknown
Siblings share ____ of alleles
about 50%
Can nuclear DNA be extracted from the red blood cells in a blood stain?
No, we need to use white blood cells for that (or mitochondrial DNA)
You inherit mitochondria ____
Maternally
Why is it important for forensics that in double stranded DNA, we always see the specific pairs A-T and G-C stuck together?
If we have one half of the double helix, we can reconstruct the other perfectly
There are a maximum of two alleles that can be found at any given locus?
No, many possible
The most common DNA reference sample is
buccal swab
Blood is ______ before collection
ALWAYS allowed to dry
DNA extraction protocol
Lyse cells open
Treat with proteinase
Remove DNA for analysis
Everyone has their own genetic footprint except
Monozygotic identical twins
We name VNTR alleles by….
how much they repeat
What are the key reasons that PCR amplification is routinely performed on all samples for modern DNA analysis?
It generates a lot of identical copies of our sample, making electrophoresis results much easier to read
It only copies the loci that we care about, cleaning up a lot of background noise
It can help compensate for damaged or degraded DNA samples by making copies of intact segments
What is the main difference between traditional genetic fingerprinting and STR analysis?
STR analysis compares the exact size of DNA fragments at specific loci instead of chopping the entire genome into fragments
Peaks in an electropherogram represent….
the alleles present
How do we calculate the likelihood of homozygous alleles?
the frequency of the allele squared
How do we calculate the likelihood of heterozygous alleles?
2 times the frequencies of each allele
How do we calculate overall likelihood?
multiply each likelihood together
CODIS steps
Amplify sample using PCR
Generate an STR profile
Verify that the profile has results at a minimum of 8 loci
Submit your data against the CODIS database
Have a DNA analyst compare the returned profile against the profile from the evidence
If the DNA analysts concludes they are from the same source, obtain a warrant for the CODIS result’s personal information.
Y-STR profiles are passed through….
father to son, and his son, and all biological sons (no daughters)
Carol and Cooper are first cousins who married. Imagine that in addition to Dot, they have a son named Daniel. How would Daniel’s genetics compare to Ben and Bob?
The three men would have the same mtDNA profiles and the same Y-STR profiles
