Exam 2 Flashcards
Master (161 cards)
1
Q
Where is DNA located in the tooth?
A
tissues in the dental pulp contain multiple cells where DNA can be isolated
2
Q
what will dental pulp do in higher temperatures?
A
it will decompose
3
Q
cementoblasts
A
cells that make the cementum
contain nuclei and mitochondria
4
Q
Ondontoblasts
A
only consist of mitochondria
5
Q
Teeth for DNA analysis
A
take DNA from the root portion of the tooth but leave the crown
6
Q
Serology
A
the study of serum or other bodily fluids
7
Q
What is blood typing?
A
evaluates the antibodies associated with different blood types
8
Q
what is forensic serology?
A
portion of forensic biology that deals with the examination and identification of biological evidence
9
Q
Forensic serology identifies what types of bodily fluids?
A
blood, semen, saliva and more
10
Q
Different bodily fluids are associated with what?
A
Different crime scenes
11
Q
Identifying bodily fluids can help link cases together or help demonstrate what?
A
possible intent
12
Q
Forensic serology does not lead to any individualization of evidence, but may lead to what?
A
an exclusion
13
Q
presumptive test
A
indicates the possibility of the presence of a specific body fluid
identify what the specimen could be
14
Q
Advantages of presumptive tests
A
rapid
sensitive
simple
often can be implemented at the scene
15
Q
Disadvantages of presumptive tests
A
not specific
16
Q
Confirmatory test
A
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
17
Q
Forensic Serology Workflow
A
Blood stain
Visual Examination
Presumptive Assay
Choice 1= Confirmatory Assay
Blood Identified
DNA Profiling
Choice 2= Species Identification
Human Blood Verified
DNA profiling
18
Q
Factors that alter decision in deciding what test to use
A
Time
money
procedures
experience
sample amount
circumstances and more
19
Q
Blood is what % of the weight of a healthy human
A
8%
20
Q
Blood is made of what two portions
A
Plasma and Cellular
21
Q
Plasma
A
55% of blood volume
22
Q
Cellular
A
red blood cells, white blood cells, and platelets
23
Q
White blood cells
A
Leukocytes
have nuclei (main source of nuclear DNA from blood)
help body fight infections
24
Q
Platelets
A
Thrombocytes
do not have nuclei
play a role in blood clotting
25
red blood cells
Erythrocytes
typical life span is 3-4 months
no nuclei
contains the protein hemoglobin
26
Hemoglobin
protein responsible for transportation of oxygen
adult hemoglobin consists of four subunits
Each subunit contains a heme moiety that binds oxygen
27
Heme molecule
ferroprotoporphyrin
28
Heme
has multiple derivatives that are utilized in testing
can bind to other chemicals like carbon monoxide
29
carbon dioxide binds to what
globular protein structure
30
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
31
presumptive blood assay tests are based on what
based on the oxidation reduction reaction catalyzed by the heme moiety of hemoglobin
32
oxidation reaction is visualized as what
a chemiluminescent, fluorescence, or change of color
33
Presumptive blood tests are often what?
oxidation reduction reactions
34
Oxidation reduction reactions
heme is the catalyst
hydrogen peroxide is the oxidant
colorless substrate (indicator)
35
If heme is present, what happens
the colorless substate is oxidized creating a colorful, chemiluminescent, fluorescent and product
36
Equation for heme?
AH(little6) (colorless) + H(little2)O Heme A(color) + 2H(little2)O
37
Colorimetric Assay
Kastle-Meyer
result is an oxidized derivative, phenolphthalein, which appears pink under alkaline conditions
37
Phenolphthalein Assay
Kastle-Meyer
38
Phenolphthalein
catalyzed by heme with hydrogen peroxide as the oxidant
39
Colorimetric Assay: Leucomalachite Green
LMG
Produces a green color
40
Leuco base form is catalyzed by heme with what as the oxidant under acidic conditions?
hydrogen peroxide
41
Leucomalachite green reduced
colorless
42
malachite green oxidized
blue-green
43
Hemastix
contains tetramethylbenzidine (TMB) and hydrogen peroxide
44
Hemastix strip
moistened and rubbed on a suspected blood stain
45
What color indicates the presence of blood in a hemastix strip?
TMB(red)= yellow
TMB(ox)= green
46
Chemiluminescent assay
emits light as a result of a chemical reaction
like a glow stick
47
Fluorescent Assay
requires exposure of the fluorescein to a certain wavelength of light
like plastic stars that fluoresce under black light
48
Advantages of assays
can be sprayed over large areas
very sensitive
in many cases does not interfere with downstream DNA applications
49
Disadvantages of assays
small stains may be diluted to a point that limits detection by the spray
50
false positive
strong oxidants= false positive
catalyze reaction in absence of heme
ex: hair coloring products, bleach
51
Any plant that contains what can catalyze an oxidation reaction leading to a false + result
a peroxidase
52
Plant peroxidases
sensitive to heat
heat will inactivate the peroxidases
53
False negatives
strong reductants= false negative
much less common than false positives
ex: lithium and zinc
54
What will form in the presence of heme molecules
crystals of certain chemicals
55
Morphology of crystals
distinctive for heme, can be compared to a known standard for confirmation
56
confirmatory tests for blood disadvantages
not as sensitive as presumptive tests
not specific for human blood
57
Heme and its Derivatives
Protoporphyrin IX
Heme
Hemochromagen
Hematin Hydroxide
58
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
59
Hematin Crystal Assay
specimens are treated with a glacial acetic acid and salts, then heated forming hematin chloride
forms a brown crystal
60
What is similar between hemochromagen and hematin crystal assays?
similar sensitivity and specificity
61
What assay performs better on aged samples?
hematin crystal assay
62
2 things that make up semen
seminal fluid and sperm cells (spermatozoa)
10^7 to 10^8 spermatozoa per mm
63
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
64
Three main features to spermatozoa
head
midpiece
the tial (flagellum)
65
Acid Phosphate used in semen screening
marker for monitoring prostate cancer
same test used in forensics
under dry conditions stable for 1 year
66
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
67
ALS
used to enhance stains
sources between 450 nm and 495 nm are used and then filtered with orange goggles
68
Ap activity is found where
prostate and other fluids like urine and fecal matter
69
AP techniques
catalyzes the removal of a phosphate group from a substrate, making a colored precipitate
70
AP techniques with Fast Blue
in the presence of, AP will react to form a purple precipitate
71
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
72
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
73
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
74
Control zone
C
Detects if the sample was loaded
to be valid, C band must appear on the test
75
Treatment zone
T
76
where Sample is loaded
S
77
Biology of saliva
water, electrolytes, proteins, antibodies, and enzymes
78
enzyme amylase
breaks down carbohydrates such as starch
79
Two major forms of human amylase
Human salivary ( HSA)
Human pancreatic (HPA)
80
Presumptive assay for saliva
ALS
Microscopic examination of epithelial cells
Starch iodine assay
phadebas reagant
81
Visual examination of epithelial cells
buccal swab
histological staining
82
Starch is present=
stay yellow
83
starch iodine test basics
dark color indicates presence of starch
no starch=amylase is present
84
Phadebas Reagent
used to detect amylase
produced in tablet form
amylase present=blue color
85
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
86
Description of RSID-Saliva test
very sensitive
human specific
rapid
deployable in the lab or in the field
87
Steps in DNA Processing
DNA Extraction
DNA quantification
DNA amplification
Electrophorese is
DNA analysis
88
Nuclear DNA
found in the nucleus and chromosomes
22 pairs or autosomal, 1 pair of sex chromosomes
Linear, long, diploid, unique
89
Mitochondrial DNA
located in the mitochondria
circular, relatively short, haploid, maternally inherited
90
method of DNA extraction choice must yield
sufficient quality
good quality DNA
high purity of DNA
91
What are you always doing in a forensic setting?
isolating total cellular DNA
92
DNA extraction techniques
cell and tissue disruption
lysis of cellular and organelle materials
removal of proteins
DNA storage
93
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
94
cell tissue and disruptions
goal is to gain access to the cells containing DNA
95
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
96
Removal of proteins
remove all and any impurities that will interfere with DNA analysis
"Washing steps"
97
Sample storage
extracted DNA needs to be stored to prevent degredation
-20 C to -80 C
98
Types of sample contamination
processing individual to sample
sample to sample
amplified DNA and Non-amplified DNA
99
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
100
methods of DNA extraction
Phenol-Chloroform (organic) Extraction(solvent based)
Chelex (boiling based)
Silica based methods
101
Proteinase
enzyme that breaks down proteins
102
removal of proteins
mixture of phenol, chloroform, and isoamylalcohol is added
Aqueous solution=water
103
DNA is precipitated out by using what
salts and ethanol
104
Pros of Organic extraction
extracts large double stranded DNA
Good quality
good yield
often considered the "gold standard" in terms of quality and yield
105
Cons of organic extraction
laborious
hazardous
multiple tube transfers( up the chances of making an error during extraction)
106
Chelex
ion-exchange resin composed of styrene divinylbenzene copoylmers
it will bind any divalent 2+ metal cations
107
DNases
nucleuses that degrade DNA
108
Chelex procedure
washing
boiling
centrifugation
109
washing step
only necessary if there is a known inhibitor for DNA amplification
higher quality=higher yielding samples
110
boiling
5-10% solution of chelex is added
incubation
leaves DNA single stranded due to heat
111
Pros of Chelex
Simple
rapid
one-tube
cheap
112
cons of chelex
single stranded DNA
very fickle
113
Silica-based method
DNA is reversibly absorbed on to silica surfaces in the presence of chaotropic salts
114
Pros of silica-based methods
high quality DNA
double stranded DNA
amenable to automation
115
Cons of silica-based methods
expensive
multiple tube transfers
116
Spermatozoa
male
117
Epithelial Cells
female
118
Pre-PCR
DNA extraction
DNA Quantification
DNA amplification
119
Post-PCR
Electrophorese is
DNA analysis
120
PCR
the exponential amplification of specific sequences of DNA
Amplified DNA=amplicon
121
DNA Structure
Deoxyribose(sugar)
Phosphate group
nitrogen base (a,t,c,g)
122
DNA strands are antiparallel
5'-3'
3'-5'
opposite of nitrogen base
123
individual nucleotides are linked by what
phospholipid bonds
124
phosphate group does what to DNA
gives it a negative charge
125
DNA bases
linked by hydrogen bonds
126
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
127
A/T; C/G are held together by what
hydrogen bonds= weak, allowing breakdown to one strand if needed
HEAT!!!!
128
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
129
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
130
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+
131
Template DNA
starting material for the reaction
multiple copies are synthesized
if quality is poor, amplification will fail
132
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
133
Polymerase
enzymes that take small subunits and bond together to form a larger molecule
134
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
135
Deoxyribonucleoside Triphosphates (dNTPs)
the new bases that will be added to the newly synthesized strands by the Taq polymerase (dATP, dTTP, dCTP, and dGTP)
136
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
137
Cycling parameters
DNA can denature and renature by changing the temperature
higher temp=single strand
lower temp=double strand
138
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
139
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)
140
- control
should not produce a DNA profile or indicate DNA being present
141
+ control
produce the expected DNA profile
if p + control does not yield the expected, all of the samples ass. are rejected
142
What does + control ensure
that the reagents and procedures work correctly
143
what does - control check for
contamination during the PCR procedure
144
DNA Quantitation
determining the amount of DNA you have after extraction
0.5 to 2.0 nanograms to the PCR reaction
"goldilocks" principle
145
What makes a good quantitation method
Human specific
accurate
validated
146
3 dif. types of DNA quantitation
slot blot procedures
intercalating dyes
quantitative PCR methods (real time PCR)
147
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
148
Intercalating Dye: PicoGreen
used to quantitate dsDNA samples
not human specific
measured with spectrophotometer
149
Quantitative PCR Assays
1. End point PCR: measures the amount of PCR product at the end of the PCR reaction
2. 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
150
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
151
End Point PCR
Analyzes the amount of DNA after PCR is completed
samples are amplified
152
Real time PCR
analyzes amount of each target after cycle
sensitive
automated
quantify different portions of DNA
detection of PCR inhibitors
153
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)
154
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
155
Electrophoresis
process in which DNA fragments are separated when placed in an electric field
relies on DNA being - charge
156
what is the matrix
physical support that holds DNA molecules
"Molecular Sieve"
agarose and polyacrylamide
157
Agarose Matrix
mixture of a solvent that is dissolved into a buffer
heated up to further dissolve and then cooled to form gel
158
Polyacrylamide matrix
cross-linking gives it an extra level of resistance over agarose
159
Differences in Matrices: Agarose
pores are large
good for separation of larger DNA fragments
160
Differences in Matrices: Polyacrylamide
pores are much smaller than agarose
can resolve 5 to 500 bp
