Exam 2 Flashcards
Master
Where is DNA located in the tooth?
tissues in the dental pulp contain multiple cells where DNA can be isolated
what will dental pulp do in higher temperatures?
it will decompose
cementoblasts
cells that make the cementum
contain nuclei and mitochondria
Ondontoblasts
only consist of mitochondria
Teeth for DNA analysis
take DNA from the root portion of the tooth but leave the crown
Serology
the study of serum or other bodily fluids
What is blood typing?
evaluates the antibodies associated with different blood types
what is forensic serology?
portion of forensic biology that deals with the examination and identification of biological evidence
Forensic serology identifies what types of bodily fluids?
blood, semen, saliva and more
Different bodily fluids are associated with what?
Different crime scenes
Identifying bodily fluids can help link cases together or help demonstrate what?
possible intent
Forensic serology does not lead to any individualization of evidence, but may lead to what?
an exclusion
presumptive test
indicates the possibility of the presence of a specific body fluid
identify what the specimen could be
Advantages of presumptive tests
rapid
sensitive
simple
often can be implemented at the scene
Disadvantages of presumptive tests
not specific
Confirmatory test
more specific and allows an investigator to say within a reasonable degree of certainty that the sample is a certain bodily fluid
identifies what the specimen is
Forensic Serology Workflow
Blood stain
Visual Examination
Presumptive Assay
Choice 1= Confirmatory Assay
Blood Identified
DNA Profiling
Choice 2= Species Identification
Human Blood Verified
DNA profiling
Factors that alter decision in deciding what test to use
Time
money
procedures
experience
sample amount
circumstances and more
Blood is what % of the weight of a healthy human
8%
Blood is made of what two portions
Plasma and Cellular
Plasma
55% of blood volume
Cellular
red blood cells, white blood cells, and platelets
White blood cells
Leukocytes
have nuclei (main source of nuclear DNA from blood)
help body fight infections
Platelets
Thrombocytes
do not have nuclei
play a role in blood clotting
red blood cells
Erythrocytes
typical life span is 3-4 months
no nuclei
contains the protein hemoglobin
Hemoglobin
protein responsible for transportation of oxygen
adult hemoglobin consists of four subunits
Each subunit contains a heme moiety that binds oxygen
Heme molecule
ferroprotoporphyrin
Heme
has multiple derivatives that are utilized in testing
can bind to other chemicals like carbon monoxide
carbon dioxide binds to what
globular protein structure
Presumptive blood assays
designed to identify traces of blood
can detect as little as 10 to the -5 power to 10 to the -6 power, fold dilutions
presumptive blood assay tests are based on what
based on the oxidation reduction reaction catalyzed by the heme moiety of hemoglobin
oxidation reaction is visualized as what
a chemiluminescent, fluorescence, or change of color
Presumptive blood tests are often what?
oxidation reduction reactions
Oxidation reduction reactions
heme is the catalyst
hydrogen peroxide is the oxidant
colorless substrate (indicator)
If heme is present, what happens
the colorless substate is oxidized creating a colorful, chemiluminescent, fluorescent and product
Equation for heme?
AH(little6) (colorless) + H(little2)O Heme A(color) + 2H(little2)O
Colorimetric Assay
Kastle-Meyer
result is an oxidized derivative, phenolphthalein, which appears pink under alkaline conditions
Phenolphthalein Assay
Kastle-Meyer
Phenolphthalein
catalyzed by heme with hydrogen peroxide as the oxidant
Colorimetric Assay: Leucomalachite Green
LMG
Produces a green color
Leuco base form is catalyzed by heme with what as the oxidant under acidic conditions?
hydrogen peroxide
Leucomalachite green reduced
colorless
malachite green oxidized
blue-green
Hemastix
contains tetramethylbenzidine (TMB) and hydrogen peroxide
Hemastix strip
moistened and rubbed on a suspected blood stain
What color indicates the presence of blood in a hemastix strip?
TMB(red)= yellow
TMB(ox)= green
Chemiluminescent assay
emits light as a result of a chemical reaction
like a glow stick
Fluorescent Assay
requires exposure of the fluorescein to a certain wavelength of light
like plastic stars that fluoresce under black light
Advantages of assays
can be sprayed over large areas
very sensitive
in many cases does not interfere with downstream DNA applications
Disadvantages of assays
small stains may be diluted to a point that limits detection by the spray
false positive
strong oxidants= false positive
catalyze reaction in absence of heme
ex: hair coloring products, bleach
Any plant that contains what can catalyze an oxidation reaction leading to a false + result
a peroxidase
Plant peroxidases
sensitive to heat
heat will inactivate the peroxidases
False negatives
strong reductants= false negative
much less common than false positives
ex: lithium and zinc
What will form in the presence of heme molecules
crystals of certain chemicals
Morphology of crystals
distinctive for heme, can be compared to a known standard for confirmation
confirmatory tests for blood disadvantages
not as sensitive as presumptive tests
not specific for human blood
Heme and its Derivatives
Protoporphyrin IX
Heme
Hemochromagen
Hematin Hydroxide
Hemocromagen Crystal Assay
stain is treated with pyridine and glucose to form crystals of pyridine ferroprotopophyrin
crystals are red
also known as Takayama crystal assay
Hematin Crystal Assay
specimens are treated with a glacial acetic acid and salts, then heated forming hematin chloride
forms a brown crystal
What is similar between hemochromagen and hematin crystal assays?
similar sensitivity and specificity
What assay performs better on aged samples?
hematin crystal assay
2 things that make up semen
seminal fluid and sperm cells (spermatozoa)
10^7 to 10^8 spermatozoa per mm
Seminal fluid is a mixture of glandular secretions
60% is seminal vesicle fluid(glow under uv light) contains flavin
30% consists of prostatic fluid secretions, contains acid phosphate and prostate-specific antigen
Three main features to spermatozoa
head
midpiece
the tial (flagellum)
Acid Phosphate used in semen screening
marker for monitoring prostate cancer
same test used in forensics
under dry conditions stable for 1 year
Prostate-Specific Antigen semen screening
used as a clinical marker for cancer
present in other tissues at lower levels
under dry conditions stable up to 3 years
ALS
used to enhance stains
sources between 450 nm and 495 nm are used and then filtered with orange goggles
Ap activity is found where
prostate and other fluids like urine and fecal matter
AP techniques
catalyzes the removal of a phosphate group from a substrate, making a colored precipitate
AP techniques with Fast Blue
in the presence of, AP will react to form a purple precipitate
Fast blue results
if color change occurs under 1 min, semen is detected
if over 1 min, means that possibly non-prostate AP is present
Christmas Tree Stain
Red Stain: Nuclear Fast Red (NFR) which stains the nuclei of the sperm
Green Stain: Picroindigocarmine (PIC) which stains the neck and tail of sperm
Identification of Prostate-Specific Antigen
immunochromatographic method
three main components: PSA antibody in the sample well, PSA antibody in the test zone, and Antiglobulin that recognizes the antibody is in the control zone
Control zone
C
Detects if the sample was loaded
to be valid, C band must appear on the test
Treatment zone
T
where Sample is loaded
S
Biology of saliva
water, electrolytes, proteins, antibodies, and enzymes
enzyme amylase
breaks down carbohydrates such as starch
Two major forms of human amylase
Human salivary ( HSA)
Human pancreatic (HPA)
Presumptive assay for saliva
ALS
Microscopic examination of epithelial cells
Starch iodine assay
phadebas reagant
Visual examination of epithelial cells
buccal swab
histological staining
Starch is present=
stay yellow
starch iodine test basics
dark color indicates presence of starch
no starch=amylase is present
Phadebas Reagent
used to detect amylase
produced in tablet form
amylase present=blue color
RSID-Saliva
immunochromatographic assay
labeled anti-HSA antibody is contained in a sample well
anti-HSA antibody immobilized onto the test zone
antiglobulin that recognizes the antibody onto the control zone
Description of RSID-Saliva test
very sensitive
human specific
rapid
deployable in the lab or in the field
Steps in DNA Processing
DNA Extraction
DNA quantification
DNA amplification
Electrophorese is
DNA analysis
Nuclear DNA
found in the nucleus and chromosomes
22 pairs or autosomal, 1 pair of sex chromosomes
Linear, long, diploid, unique
Mitochondrial DNA
located in the mitochondria
circular, relatively short, haploid, maternally inherited
method of DNA extraction choice must yield
sufficient quality
good quality DNA
high purity of DNA
What are you always doing in a forensic setting?
isolating total cellular DNA
DNA extraction techniques
cell and tissue disruption
lysis of cellular and organelle materials
removal of proteins
DNA storage
the animal cell
open cell membrane to gain access to nucleus
break down nuclear membrane
DNA is encapsulated in proteins, helps protect it
break down chaperone proteins and purify DNA in an environment that protects it
cell tissue and disruptions
goal is to gain access to the cells containing DNA
Lysis of Cellular and organic membranes
one cells are accessible, need to disrupt the cell membranes, organelles, and proteins
Lysis is performed with salts, detergents (SDS) and enzymes heat
needs to be performed in a buffer
Removal of proteins
remove all and any impurities that will interfere with DNA analysis
“Washing steps”
Sample storage
extracted DNA needs to be stored to prevent degredation
-20 C to -80 C
Types of sample contamination
processing individual to sample
sample to sample
amplified DNA and Non-amplified DNA
Systematic Checks
use of a pre- and post PCR room
separate processing of evidence and reference samples
use of DNA free-reagents
everyone in the lab submits a DNA sample
methods of DNA extraction
Phenol-Chloroform (organic) Extraction(solvent based)
Chelex (boiling based)
Silica based methods
Proteinase
enzyme that breaks down proteins
removal of proteins
mixture of phenol, chloroform, and isoamylalcohol is added
Aqueous solution=water
DNA is precipitated out by using what
salts and ethanol
Pros of Organic extraction
extracts large double stranded DNA
Good quality
good yield
often considered the “gold standard” in terms of quality and yield
Cons of organic extraction
laborious
hazardous
multiple tube transfers( up the chances of making an error during extraction)
Chelex
ion-exchange resin composed of styrene divinylbenzene copoylmers
it will bind any divalent 2+ metal cations
DNases
nucleuses that degrade DNA
Chelex procedure
washing
boiling
centrifugation
washing step
only necessary if there is a known inhibitor for DNA amplification
higher quality=higher yielding samples
boiling
5-10% solution of chelex is added
incubation
leaves DNA single stranded due to heat
Pros of Chelex
Simple
rapid
one-tube
cheap
cons of chelex
single stranded DNA
very fickle
Silica-based method
DNA is reversibly absorbed on to silica surfaces in the presence of chaotropic salts
Pros of silica-based methods
high quality DNA
double stranded DNA
amenable to automation
Cons of silica-based methods
expensive
multiple tube transfers
Spermatozoa
male
Epithelial Cells
female
Pre-PCR
DNA extraction
DNA Quantification
DNA amplification
Post-PCR
Electrophorese is
DNA analysis
PCR
the exponential amplification of specific sequences of DNA
Amplified DNA=amplicon
DNA Structure
Deoxyribose(sugar)
Phosphate group
nitrogen base (a,t,c,g)
DNA strands are antiparallel
5’-3’
3’-5’
opposite of nitrogen base
individual nucleotides are linked by what
phospholipid bonds
phosphate group does what to DNA
gives it a negative charge
DNA bases
linked by hydrogen bonds
Characteristics of DNA
double stranded
synthesized (made) in the 5’ to 3’ direction
each strand is antiparallel to opposite strand
each strand is complementary to its adjacent strand
A/T; C/G are held together by what
hydrogen bonds= weak, allowing breakdown to one strand if needed
HEAT!!!!
2 requirements of renaturation of DNA
Must have charged molecules (salts) to neutralize the - charge of DNA
A temperature high enough to disrupt random base pairing, but low enough to allow the strands to come together
Basics of PCR
Works predominantly, can go to one strand back to two strands
when denatured, 2 single strands can be used as templates for the creation of new strands
5 important components for DNA amplifications
Template DNA: Starting material
Primers: small fragments of DNA, allows amplification to be specific
Taq Polymerase: enzyme can add bases to new strand
Deoxyribonucleoside triphosphates (dNTP’s): bases added by taq polymerase enzyme
Cofactors (divalent cations): required for the enzymatic activity of the polymerase; ex: mg2+
Template DNA
starting material for the reaction
multiple copies are synthesized
if quality is poor, amplification will fail
Primers
18-25 nucleotides of DNA attach to the denatured strands
flank the region of interest
only attach to specific region
allows for amplification of specific region of DNA
Polymerase
enzymes that take small subunits and bond together to form a larger molecule
Taq
adds the dNTPs to the newly synthesized DNA
special polymerase isolated from the bacterium Thermicus aquaticus
work at a range of temp. including very high ones
moves down the strand (5’ to 3’) adding the complementary base
Deoxyribonucleoside Triphosphates (dNTPs)
the new bases that will be added to the newly synthesized strands by the Taq polymerase (dATP, dTTP, dCTP, and dGTP)
Cofactors
required by Taq Polymerase to be activated
same principle as discussed with nucleuses, instead this is an enzyme you want to work in the amplification process
Cycling parameters
DNA can denature and renature by changing the temperature
higher temp=single strand
lower temp=double strand
3 different cycles that are repeated
Denaturing (94-95 C) : double strand=single strand
Annealing (50-60 C): primers attach to single strand DNA
Extension (~72 C) : Taq polymerase adds new bases to new strand
Quality control for PCR
very sensitive
procedures
pre-PCR and post-PCR
PPE needs to be worn
reactions are performed in a chemical hood
1. - control (all reagents but no DNA)
2. + control (known DNA sample)
- control
should not produce a DNA profile or indicate DNA being present
+ control
produce the expected DNA profile
if p + control does not yield the expected, all of the samples ass. are rejected
What does + control ensure
that the reagents and procedures work correctly
what does - control check for
contamination during the PCR procedure
DNA Quantitation
determining the amount of DNA you have after extraction
0.5 to 2.0 nanograms to the PCR reaction
“goldilocks” principle
What makes a good quantitation method
Human specific
accurate
validated
3 dif. types of DNA quantitation
slot blot procedures
intercalating dyes
quantitative PCR methods (real time PCR)
Intercalating Dyes
fits in between two parts
certain dyes that intercalate between base pairs of DNA without breaking the double stranded structure
dyes are fluorescently labeled
Intercalating Dye: PicoGreen
used to quantitate dsDNA samples
not human specific
measured with spectrophotometer
Quantitative PCR Assays
- End point PCR: measures the amount of PCR product at the end of the PCR reaction
- Real-time PCR: measures the amount of PCR product as the reaction is occurring; often measured during the exponential phase of the reaction
advantageous because it allows for the detection of inhibitors which provides more information prior to further processing
Different Phases of PCR
4 phases
Lag: beginning. amt of amplification is slow
Exponential: number of amplicons increase a lot
Linear: Amplification begins to slow due to reagents become limited
Plateau: no more amplification, reagents no longer available
End Point PCR
Analyzes the amount of DNA after PCR is completed
samples are amplified
Real time PCR
analyzes amount of each target after cycle
sensitive
automated
quantify different portions of DNA
detection of PCR inhibitors
TaqMan Method
uses a probe that binds within the amplified region of DNA
Binding happens after double stranded DNA has become single stranded
probe has a reporter dye (R) and a quencher dye (Q)
How TaqMan Works
if separated, Reporter will fluoresce
5’ exonuclease activity
during extension phase, taq cuts off the reporter allowing it to move away from the quencher and fluoresce
Electrophoresis
process in which DNA fragments are separated when placed in an electric field
relies on DNA being - charge
what is the matrix
physical support that holds DNA molecules
“Molecular Sieve”
agarose and polyacrylamide
Agarose Matrix
mixture of a solvent that is dissolved into a buffer
heated up to further dissolve and then cooled to form gel
Polyacrylamide matrix
cross-linking gives it an extra level of resistance over agarose
Differences in Matrices: Agarose
pores are large
good for separation of larger DNA fragments
Differences in Matrices: Polyacrylamide
pores are much smaller than agarose
can resolve 5 to 500 bp
