Technical Oral Study Guide Flashcards
1
Q
what is screening?
A
the examination and sampling of evidence for DNA analysis and/or preservation
the first step of the DNA analysis process
2
Q
what is serology?
A
the testing and identification of bodily fluids (blood and seminal fluid)
3
Q
what are the two serological tests that LSPCL uses for blood?
A
phenolphthalein (PHE) or Kastle-Meyer Test
ABAcard Hematrace
4
Q
explain the PHE test and how it’s performed at LSPCL. are there any limitations to this test?
A
redox reaction
hemoglobin (Hb; from blood) loses two electrons and is the catalyst in breaking apart H2O2 (hydrogen peroxide) into two H2O (water) molecules
phenolphthalin donates two electrons back to Hb, so Hb remains unchanged, and phenolphthalin is oxidized into phenolphthalein resulting in the pink color change
- phenolphthalin is kept in its reduced state by adding zinc to the reagent bottle
add 1-2 drops of PHE to small cutting/swab. wait 30 seconds, then add 1-2 drops of H2O2. an immediate color change to pink will occur if the sample is presumptive positive for blood. no color change after 15 seconds = negative
NOTE: all samples will turn pink over time because of normal oxidation
limitation: not human specific
5
Q
explain the hematrace test and how it’s performed at LSPCL. are there any limitations to this test?
A
6
Q
what are the three presumptive serological tests at LSPCL used for semen?
A
7
Q
explain the acid phosphatase (AP) test and how it’s performed at LSPCL. are there any limitations to this test?
A
8
Q
explain the ABAcard p30 test and how it’s performed at LSPCL. are there any limitations to this test?
A
9
Q
what is the only confirmatory test we use at LSPCL for semen? explain the test (dyes, etc) and why it is considered confirmatory. are there any limitations?
A
10
Q
what is DNA extraction and what are the two goals?
A
isolation and purification of DNA
two goals: maximize the amount of high quality DNA recovered by removing inhibitors and removing nucleases that promote the breakdown of DNA & separate DNA from other cellular components
11
Q
how much DNA can be recovered from a single diploid/haploid cell?
A
~6 pg/diploid cell
~3 pg/haploid cell
12
Q
what are the two extraction methods we use at LSPCL?
A
- solid phase: silica coated magnetic beads
- differential extraction
13
Q
explain the 4 basic steps of solid phase extraction
A
- lyse – cells are lysed using a tissue lysis buffer (ATL, G2, MTL) with pK & DTT added to break down histones and allow the DNA to be unwound
- bind – DNA becomes attracted to the silica beads due to the low pH and high chaotropic salt concentrations
- wash – the bound DNA is washed with a wash buffer ensuring the removal of contaminants and proteins
- elute – concentrated and purified DNA is eluted in TE buffer; it is eluted into high pH and low chaotropic salt concentrations
14
Q
how does the DNA become bound to the silica beads?
A
chaotropic salts are introduced that lower the pH of the solution
the low pH causes the DNA to become (-) charged and the beads (+) charged
the DNA is then attracted to the beads
once the salts are washed away, the pH raises up again, which causes the DNA to become eluted
15
Q
what reagent can be added to samples with suspected low amounts of DNA during extraction to increase the recovery yield and why?
A
carrier RNA (cRNA) – it drives the binding process of the DNA to the silica beads by adding nucleic acids
16
Q
list the 6 reagents used during extraction and briefly describe their purposes
A
- ATL buffer – tissue lysis buffer. Aids in cell lysis so the DNA is exposed
- G2 buffer – tissue lysis buffer, but is gentler than ATL. Used in differential extractions to lyse the epithelial cells first.
- proteinase K (pK) – serine protease that digests proteins imbedded in the cell membranes, inactivates nucleases that break down DNA, and breaks down histone proteins allowing the DNA to unwind
- dithiothreitol (DTT) – breaks down the disulfide bonds of cell membranes allowing the release of DNA. Especially crucial for digesting hair & sperm cells. Additional to help lyse
- MTL buffer – added by EZ2 instrument to large volume extractions. Lysis buffer with chaotropic salts
- carrier RNA (cRNA) – enhances binding of DNA to surface of magnetic beads, especially helpful when low amounts of DNA are expected. Introduces extra nucleic acids. Added to all evidence samples, references that are expected to be low quality DNA (dead bodies, etc.) but if you add to one reference, you must add to all references on protocol + the EB
17
Q
what is a differential extraction and how does it work?
A
the goal is to separate two cell types, in our case, sperm cells and epithelial cells
procedure –
Qiacube
- non-sperm cell lysis by adding G2 and pK and incubate (fraction 1, F1; epi cells)
- centrifuge: supernatant contains free DNA from epi cells; sperm pelleted at bottom (still intact)
- remove supernatant without disturbing sperm pellet = F1
- sperm cell lysis by adding ATL, pK, DTT fraction 2, F2)
- add cRNA before purification
- purification via EZ2
18
Q
what are inhibitors? what are three basic types of inhibitors (list a few examples of each)? how do we overcome them?
A
inhibitors are compounds that impede the PCR rxn by interfering with the reaction between DNA and Taq polymerase
can co-extract with DNA and cause partial profiles or no DNA results
3 main types –
1. internal inhibitors: found in bodily fluids
a. Heme in blood
b. Bacteria in vaginal/fecal samples
c. Calcium in bone/teeth
d. Urea in urine
e. Spermine and spermidine in semen
2. substrate inhibitors: found in the environment
a. Textile dyes (indigo dyes in denim)
b. Tannic acid in leather
c. Humic acid in soil
d. Organic compounds in food (calcium in milk)
3. inhibitors from extraction process
a. Chaotropic salts
b. Silica beads
c. Detergents (ATL, MTL, G2)
d. Proteases (pK)
to overcome inhibitors –
EZ2 purification is extremely efficient at removing inhibitors
real time PCR quantification kits assess inhibition through use of an internal PCR control (IPC)
diluting the sample to dilute inhibitors, but it will also dilute the DNA
19
Q
what are controls?
A
samples used to demonstrate a method works correctly & to ensure data are valid
they are utilized throughout the whole DNA process
we have positive and negative controls
20
Q
briefly describe the three controls we use at LSPCL
A
- extraction blank (EB) – detects contamination of extraction reagents (introduced at the beginning; extraction); each ext. set utilizes an EB; follows the samples through the whole process
- positive amplification control (PC) - introduced at amplification; known DNA profile to ensure all processes worked properly
- negative amplification control (AB) – amplification blank; introduced at amplification; no DNA profile, same as the EB - can detect contaminants
21
Q
what is quantification and what are the three reasons we do it?
A
quantification is determining the quantity and quality of DNA; this process is used to make downstream processing decisions
3 reasons:
1. determining the quantity and quality of the human DNA available for amplification (important for data analysis)
2. we need to hit the optimized target range for DNA amplification (1ng) because too much or too little DNA will affect the electropherogram results
3. FBI QAS requirement – standard 9.4
22
Q
briefly explain TaqMan chemistry in qPCR
A
utilizes a TaqMan probe (the probe has a reporter dye on the 5’ end and a quencher on the 3’ end) that hybridizes to complementary targets on the DNA strand between the forward and reverse primers
when the reporter dye and the quencher are in close proximity, the quencher causes suppression of the fluorescence of the reporter
Taq polyermase has 5’-3’ exonuclease activity which cleaves only the probes that have annealed to the target sequence and as the reporter disassociates from the quencher, it results in an increase of fluorescence from the reporter allowing for quantitative measurements of the accumulation of the product
23
Q
describe the PowerQuant kit we utilize at LSPCL
A
it is a 5-dye, 4-target hydrolysis probe-based qPCR multiplex that amplifies multicopy targets to quantify the total human & human male DNA present in a sample (also detects degradation/inhibition)
it measures fluorescence over time (the more DNA = the more fluorescence)
4 targets –
1. autosomal DNA: 84 bases
2. Y DNA: 81 bases & 136 bases
3. degradation: 294 bases
4. inhibition: 435 bases
24
Q
describe the 4 phases of qPCR
A
- lag phase – baseline; measures background noise; before significant product formation; PCR product is increasing but fluorescence is too low to be detected
- exponential phase – close to, if not 100% efficiency, amplicon formation is doubling each cycle since the PCR components are in excess; optimal place to measure fluorescence
- linear phase – reaction efficiency slows to an arithmetic increase; PCR components start to fall below critical concentration causing the amplification efficiency to slow
- plateau phase – product formation has diminished; PCR components have been exhausted and reached the end of effectiveness
25
what is the Ct/q value?
cycle threshold/ quantification cycle – cycle at which fluorescence exceeds a threshold (cycle threshold) for baseline noise
it is inversely proportional to amount of starting template DNA
low Cq value = less cycles for fluorescence = more DNA
high Cq value = more cycles for fluorescence = less DNA
the software calculates the DNA concentration based on the CT/q value of the sample and comparing it to the standard curve CT/q’s
26
explain the standard curve for qPCR
the standard curve is a graph of the Ct/q of the standards vs. the Log of the concentration of the standards
there are four standards - the values are collected during the exponential phase of the PCR reaction
a regression line is calculated using the best fit curve with the quantification standard data points
regression line formula: Ct/q = m(LogQTY) + b
27
what are the concentrations of the standards used at LSPCL for qPCR and how do we make them?
1. 50ng/ul = 50µl undiluted male standard only
2. 2ng/ul = 4ul undiluted male standard + 96ul dilution buffer
3. 0.08ng/ul = 4µl of 2ng/µl standard + 96µl dilution buffer
4. 0.0032 ng/ul = 4µl of 0.08ng/µl standard + 96µl dilution buffer
NTC = 50µl dilution buffer
28
what values do you need to assess from the standard curve?
1. R2 value is a measure of the closeness of fit between the standard curve regression line and the individual CT/q data points of the standards
1 is perfect; >0.99 is close fit
2. slope indicates the PCR amplification efficiency for the assay
-3.3 indicates 100% efficiency; an acceptable slope range is -3.1 to -3.6
3. y-intercept indicates the expected CT/q value for a sample with a quantity of 1ng/ul; in other words, it is the y value when the x value equals 0
29
what is the IPC in qPCR?
it is a novel DNA sequence included in the primer mix that provides confirmation that all assay components are functioning as expected and confirm the validity of negative results while providing the ability to determine if the sample is inhibited
evaluation occurs by observing a potential shift in the IPC value greater than what has been established through a lab’s validation (LSPCL ≥ 0.3)
30
what are the 4 components in the PQ kit?
PQ 2X master mix
PQ 2X primer/probe/IPC mix
water, amplification grade
CXR dye
31
what is CXR dye?
a passive reference dye included in the PCR master mix and it is present in all wells of the reaction plate at the same concentration
our software normalizes each reporter signal by dividing it by the fluorescent signal of the passive reference dye allowing the software to account for variations in signal caused by pipetting inaccuracies/instrument fluctuations and make better well-to-well comparisons of the reporter signal
32
describe how fluorescence is detected on the QS5
sample wells are illuminated with a bright white light emitting diode (LED)
light first passes through five excitation filters and the optical adhesive cover before reaching the sample wells
the fluorescent dyes are excited and pass through five emission filters which are then detected by a CMOS (Complementary Metal Oxide Semiconductor) camera
the fluorescent dyes used have a λ in 500nm - 700nm range; depending on the wavelength, filters separate light across detectors
the electrical signal is then read by the software with the background fluorescence being removed
33
at LSPCL, how many cycles of qPCR are there for quant?
39 cycles
34
what is amplification and what are the three purposes?
the process of making copies of the target DNA sequences
3 purposes –
1. target specific regions of DNA
2. generate millions of copies of the target regions
3. needed to detect STR fragments during capillary electrophoresis by tagging the target sequences with fluorescent dyes
35
what are STRs?
short tandem repeats (aka microsatellites) - short DNA sequences that are repeated several times in a row
they are small polymorphic regions, made up of 2-6 base pair repeat units, which allows for multiplexing and is compatible with degraded DNA and the number of repeats differ between individuals and various populations
36
what are the 4 various repeating structures found in STRs?
1. simple repeats - consist of one repeating sequence of identical length and sequence; some can have non-consensus (microvariant) alleles such as 9.3
2. compound repeats - consists of repeating sequence of identical length but a variable sequence
3. complex repeats - consists repeats of variable length as well as variable sequences
4. complex hypervariable repeats - have numerous non-consensus alleles that differ in size and sequence
37
what is the most common STR marker found in Fusion 6C?
tetra-nucleotides (4 base pair repeat units)
38
what is PCR? describe the basic steps
polymerase chain reaction – a process by which specific regions of DNA can be replicated to yield multiple copies
a small amount can be copied in large quantities over a short period of time
3 steps –
1. denaturation: the two DNA strands are denatured or separated by heat
2. annealing: the sample is then cooled to allow the primers to anneal to the DNA segments
3. extension: the temperature is raised to allow the DNA polymerase to add nucleotides to extend the primers to produce a copy of each DNA template strand
- final extension - adenylation
39
describe the PCR thermal cycling steps used at LSPCL for Fusion 6C
incubation – 96*C for ~1 minute
29 PCR cycles
- denature – 96*C for ~5 seconds
- anneal/extension – 60*C for ~1 minute
final extension – 60*C for ~10 minutes
40
what does multiplexing mean?
it's when more than one region is copied simultaneously by adding more than one primer set to the reaction
with multiplexes, the primer design, components, and thermal cycling parameters need to be optimized
40
what is another name for PCR products and at what cycle does doubling of the product theoretically start and why?
amplicons
3rd cycle
- the first cycle produces an amplicon starting with the primer and everything afterward; meaning more is copied than just the target sequence
- the second cycle produces two amplicons of only the target sequence; however, only the one with the forward primer will be detected since it is the only primer tagged with a fluorescence dye
- at the beginning of third cycle, you now have one sequence of only the targeted area with the probe and at the end of the third cycle you will have two with the tagged fluorescence dye
41
how many loci are amplified with the F6C kit?
27 total loci
23 autosomal & 4 sex determining
42
why are primers added in higher concentrations of DNA to the amplification rxn?
primers are added in higher concentrations to help drive the PCR rxn forward
43
what are primer dimers?
when primers bind and interact with each other
44
what are the 8 components that are required in PCR reactions?
1. DNA template
2. primers
3. DNA (Taq) polymerase
4. bovine serum albumin (BSA)
5. potassium chloride
6. magnesium chloride
7. deoxynucleoside triphosphates (dNTPs)
8. buffer Tris-HCl
45
what are dNTPs?
building blocks used to create new replicate DNA strand
four synthetic nucleotides consisting of a base bonded to a sugar bonded to a phosphate group - dATP (adenine), dCTP (cytosine), dGTP (guanine), dTTP (thymine)
dNTP complementary to bases are added one by one
lower concentration minimizes mispairing and reduces the likelihood of extending misincorporated (mix-matched bases; promotes chain termination) nucleotides
46
what does BSA do during a PCR rxn?
protein added to help stabilize polymerase
reduces/prevents PCR inhibition
47
what does potassium chloride do during a PCR rxn?
neutralizes the charge present on the backbone of DNA
facilitates annealing of the primer to the template DNA
reduces the repulsion between the negatively charged DNA strands (primers and templates) thereby stabilizing the primer template binding
48
what is the purpose of Taq polymerase and what modification was made to prevent non-specific products from forming at setup? where does Taq polymerase derive from?
Taq polymerase incorporates dNTPs, but it lacks 3’ to 5’ exonuclease
it has been chemically modified to be inactive until the reaction reaches ~96*C for 10-11 minutes (AKA Hot Start PCR) improving the specificity, sensitivity, and yield of PCR and allows for room temp setup
at room temp, the primers can anneal non-specifically to the template DNA and unmodified Taq does exhibit some activity at this temp and can cause non-specific amplicons
Thermus aquaticus
48
what does magnesium chloride do during a PCR rxn?
co-factor required by DNA polymerase to function
aids in primer annealing
increasing the concentration increases yield, but decreases specificity and fidelity
49
what is adenylation?
Taq polymerase tends to add a single nucleotide (dATP) to the blunt end of a DNA fragment, creating a single nucleotide overhang at the 3’ end of the amplicon
if adenylation does not occur for some amplicons, you will see a small peak that is 1 bp shorter than the true peak; this is referred to as “-a” peak
50
why are free magnesium and potassium chloride needed in the amplification reaction?
magnesium is required for Taq to function and is also needed to bind to the template DNA, primers, and dNTPs
KCl facilitates the annealing of the primer to the template DNA by neutralizing the charge present on the backbone of DNA reducing the repulsion between the negatively charged DNA strands (both template and primers)
50
what is the purpose of BSA and Buffer Tris-HCl in the amplification master mix?
BSA is added to help stabilize the polymerase and can help reduce inhibition
buffer Tris-HCl is added to stabilize the pH since it can alter the fidelity of a dNTP insertion
51
what is the most common method for tagging amplicons and why do we tag them?
dye-labeled primer insertion is the most common method for forensic STR DNA analysis; the dyes are attached to one primer (forward) in a pair used to amplify an STR marker
dye labeled primers are incorporated during the multiplex PCR amplification giving a specific color tag to each amplicon; these amplicons are then distinguished using a CCD camera on a capillary electrophoresis (CE) instrument
51
what sizing standard do we use?
WEN ILS 500 - there are 21 DNA fragments and they are each labeled with the WEN dye (which is orange)
52
how many dyes are used in F6C kit?
6 dyes - blue, green, yellow, red, purple, orange
53
what is electrophoresis and what are the two purposes of it?
electrophoresis – movement of charged molecules through a medium in an electric field
primary methodology used for separating and detecting DNA fragments
separating - 51 dye-labeled DNA fragments per contributor; migration speed represents size of DNA fragments
detecting - dye detected and size indicates corresponding marker; size and marker are used to determine allele designation by analysis software; allele combinations designate genotype
53
what are the two types of electrophoresis and which do we utilize at LSPCL? what are three reasons as to why we utilize this method over the other?
gel electrophoresis & capillary electrophoresis
1. very small quantities of samples are consumed in the injection process
2. no extra steps such as scanning the gel or taking a picture required
3. no cross contamination from samples leaking over adjacent walls
54
briefly describe the CE process
samples are prepared in a mixture of formamide and an internal size standard
injection of the samples into the instrument is performed via an electrokinetic injection using low voltage for a few seconds (LSPCL currently uses 18 second injection time)
separation occurs in the capillaries utilizing platinum electrodes that act as the cathode, POP 4 polymer (Performance Optimized Polymer -4) that acts as the sieve, buffers, and denaturants
detection occurs through the excitation of the fluorescent dyes using lasers and documenting the emission traits on a CCD camera (Charged-Coupled Device) camera with defined virtual filters
data analysis is performed using the data from the detector that is measured in Relative Fluorescence Units (RFUs)
- rfus are directly proportional to the relative quantity of DNA in the sample
55
what are the three requirements for STR typing using capillary electrophoresis?
spatial resolution, spectral resolution, DNA sizing precision
56
why is spatial resolution required during STR typing?
it maps the position of each capillary to a region on CCD camera and is used to determine which capillary a particular signal corresponds with
is needed when the capillary is replaced, the detector door is opened or detection cell moved, or when the entire instrument is moved
separates STR alleles that differ in size by one nucleotide such as with variant alleles
57
why is spectral resolution required during STR typing?
it is necessary for the software to separate out spectral dye overlap of fluorescent dyes; the software will analyze the data from each of the dyes and creates a matrix or spectral calibration file for each capillary
is needed after an array change, when service is performed on optics, or when excessive pull-up is present in data; if calibration doesn’t work, then the baseline can be uneven or pull-up can occur
separates fluorescent dye colors from one another so that PCR products labeled with different dyes can be resolved
58
why is DNA sizing precision required during STR typing?
ensured with an internal sizing standard used to calibrate each run so that samples can be related to allelic ladders
58
what purpose does formamide serve and why must we use high quality formamide (Hi-Di)?
deionized formamide helps keep the DNA denatured while reducing the salt levels (chloride) and aids in the electrokinetic injection process
Hi-Di formamide has a low conductivity; however, as it breaks down, it produces negatively charged formic acid which are preferentially injected into the capillary instead of the DNA
59
explain electrokinetic injection
in a multicapillary array, the cathode surrounds each capillary and an electric voltage is applied while the end of the capillary is immersed into the liquid DNA sample
a flow of current is generated by the applied voltage which causes the negatively charged DNA fragments to move into the end of the capillary because the DNA is repelled from the negatively charged cathode that has been inserted into the sample well; the DNA is also being attracted to the positively charged anode at the opposite end of the system
during the injection, other negatively charged salt ions such as chloride, will compete with the DNA to get into the capillary
60
since all DNA segments have a uniform negative charge and are solely separated based on the size of the fragment with smaller segments moving faster, what are 5 components that impact DNA separation? briefly describe how they impact a separation.
1. capillary array/capillaries - narrow glass tubes of fused silica with an outer polyimide coating that provide physical strength/durability while allowing flexibility of silica tube
2. polymer - POP-4 (performance optimized polymer 4%); slows DNA for detection of alleles differing by a single base; permits allele sizing (call) with a really precise measurement; contains high concentration of urea which helps keep the DNA denatured
3. buffer - upplies charge carriers (ions) for conducting electrophoretic current across the capillary; stabilizes and solubilizes the DNA; if not properly replenished, the current can fluctuate affected the DNA separation
4. temperature - needs to be stable to ensure migration pace remains constant; post-amp room is usually 23-24*C; too cold – DNA moves slower; too warm – DNA move faster
5. voltage - need constant voltage during and between every run
61
explain how DNA moves through the capillaries and how it is detected
DNA moves through the capillary with the electrophoretic flow from the cathode (negative) to the anode (positive)
there is also the electroosmotic flow that is flowing in the opposite direction of the electrophoretic flow
during electrophoresis, anode buffer is also being pulled into the capillaries and it lines the negatively charged silica glass capillaries. It slowly moves toward the negatively charged cathode and works against the electrophoretic flow to help slow the movement of DNA along with the polymer
there is a defined area of the capillary that is used as the detector window; as the dyes pass through through this area, they are excited by the solid state laser and fluoresce
optical filters are then used to separate the fluorescent signal based on wavelength of the dye
the CCD camera measures the light intensity emitted and converts to an electronic signal (RFUs)
different wavelengths of signal are detected on a different locations of the camera due to diffraction grating; the location of light detected on the camera determines the color with the locations being defined by virtual filters
62
what is the difference between the electrophoretic flow (EPF) and electroosmotic flow (EOS)?
EPF - DNA moves through capillary from the cathode (-) to the anode (+)
EOF - opposite of the electrophoretic flow; movement of the anode (+) buffer toward the cathode (-) buffer; polymer reduces (slows) “backward” flow so DNA can still move toward the positive charge
63
what does POP-4 contain that helps keep DNA in a denatured state?
it contains a high concentration of urea which helps keep the DNA denatured
64
why is it important for all the wells in an inject to have at least master mix or formamide in them?
capillaries must not be allowed to dry out. If the inlet or outlet do, then urea or salts from the buffer will form crystals that clog the narrow openings which can result in low or no current when the voltage is applied
the end of the capillaries should be stored in deionized water or buffer to prevent drying out
65
what are the two components of the CE master mix?
formamide and WEN
9.5 ul Hi-DI + 0.5 ul WEN
66
during CE, how is the data from the detector measured?
during CE, data from the detector is measured in time and intensity
the detected DNA fragments are represented as peaks on the egram with an X and Y axis
X axis – time when the fragment was detected; correlates to base pair size (migration speed)
Y axis – intensity/height of peak (RFUs); correlates to amount of DNA present
the instrument output file (.hid) is imported into GeneMapper and converts raw data to a readable form (egram)
67
what are the 4 steps in data review?
1. view raw data for each ladder, control, and sample checking for primer peaks
2. apply analysis settings and analyze project
3. assess the internal size standards, allelic ladders, positive controls, negative controls, and reagent blanks
4. assess each sample for presence of extraneous peaks and determine if they may interfere with interpretation process
68
describe the Local Suuthern Method
GMID-X uses an internal lane standard (ILS) that is included with each sample well
the ILS contains fragments spanning the analysis size range and a sizing curve for each sample is generated from these points
the size of the samples DNA fragments are sized by comparing their migration time with those of the ILS and the sizing algorithm used to determine the size of the allele is the Local Southern Method
a curve is created with three standard points, two point below and one point above the fragment, then a fragment size is determined
another curve is created using an additional set of three points, this time one point below and two points above the fragment, to assign another value
finally, the two curves are averaged to determine the unknown fragment length
after determining the size of the peak, it is then compared to the allelic ladder and placed into a bin and given the allele call for that bin
69
describe the analytical threshold
defines the height requirement at and above which detected peaks can be reliably distinguished from background noise
data analysis begins with the analytical threshold to evaluate general sample peak heights and baseline noise for increased activity or pull-up
100rfu
70
describe the saturation threshold
an upper RFU threshold which should only be applied to a sample exhibiting high peak heights where baseline noise or pull-up is present above the analytical threshold causing artifacts to be labeled as a true peak
300rfu
71
describe the stochastic threshold
the peak height or signal magnitude below which it is reasonable to assume that allelic dropout of a sister allele in a heterozygous pair may have occurred
350rfu
72
what is the difference between biological and technological artifacts? give examples of each
biological artifacts – product of PCR amplification - stutter, -A/split peaks/non-template nucleotide addition, non-specific amplification
technological artifacts – product of an instrumentation (technology) issue - migration issues, electronic spikes, dye blobs, formamide blobs, pull-up peaks, size standard failure
73
what is migration? how do you resolve?
DNA fragments migrating at different rates between injections on the same run/plate
commonly caused by fluctuations in temperature
to resolve – use a different ladder; if still present, reinject affected samples, a ladder, & PC
74
what are spikes? how do you resolve?
caused by an electrical impulse during injection
most often, present in more than one dye channel
to resolve – reinjection; not reproducible
75
what is –A/split peaks? how do you resolve?
-A peaks occur when PCR ends before the completion of the last stage, which is adenylation (this is when Taq Polymerase will add an extra adenosine to the 3’ end of the amplicon)
this causes a peak that will be 1 base pair shorter than the true allele and most often occurs in samples with too much template DNA
to resolve – place the amp plate back on the thermal cycler for an additional 60 degree incubation period for Taq to finish adenylation
75
what is stutter? how do you resolve?
caused by strand slippage of the Taq polymerase during PCR extension when the primer-template complex becomes unpaired
minor peak typically observed one repeat unit shorter than the primary allele (in rare instances, it is one repeat unit longer than the primary allele)
common, well-recognized artifact and is typically < 20% of parent peak in reverse position and ~3% in forward position
GMID-X contains stutter filters
to resolve – if stutter is suspected, it should be calculated and documented on egram
75
what are the 7 steps of interpretation?
1. interpretable vs. uninterpretable
2. minimum number of contributors and evaluate sex determination
3. distinguishable or indistinguishable
4. designate genotype(s) is applicable
5. utilize available assumptions for further resolution of genotypes if appropriate
6. compare to applicable reference samples
7. perform statistical evaluation when applicable
75
what is loss of resolution? how do you resolve?
loss of resolution is when peaks shorten and broaden as you move to the right of the e-gram
over-saturation of CCD camera caused by off-scale data
to resolve - in some cases with a re-inject, but if it is caused by the breakdown from an old plate then a new CE plate will have to be created
75
what is data interpretation and what is the main purpose of interpretation? what does the evaluation include (what are you looking for)?
data interpretation is the evaluation of the DNA results obtained from items of evidence
the primary goal of interpretation is to determine the possible genotype combinations of the contributor(s)
evaluation includes:
- distinguishing between alleles and artifacts
- determination if a profile is single source or a mixture
- determination of number of contributors
- attempts to separate a mixture into possible donors
- comparing known reference standards to profiles obtained from questioned samples
interpretations are preformed based on the laboratory’s procedures
75
what is pull up? how do you resolve?
pill-up represents failure of the analysis software to discriminate between different dye colors caused by oversaturated data which causes the dyes to bleed over or pull-up into another dye channel
to resolve – raise to saturation threshold in GMID-X (300rfu)
if there is excessive pull-up occurring at the saturation threshold and is interfering with interpretation, then the sample can be re-amped using less template DNA
75
according to the FBI QAS, what designates you as an analyst?
an analyst can conduct and/or direct the analysis of forensic samples, interpret data, reach conclusions, and generate reports
75
what is carry-over? how do you resolve?
carry-over occurs when a sample from the previous injection transfers over into the next injection
typical cause is a highly or over-concentrated sample
peak morphology looks real typically, peaks are within bins
real DNA that “carried over” from one sample to another sample injected in same capillary
to resolve - a simple fix would be to inject blanks then re-inject sample
76
what is the PHR of sister alleles in a robust single source sample?
>/= 70%
76
what is a DNA mixture?
mixtures arise when more than one individual contributes DNA to a sample
accurately determining the number of contributors is critical
76
what is cross-talk? how do you resolve?
crosstalk is when there is some signal transference from one capillary into another directly adjacent to it
typical cause is a highly or over-concentrated ample
peak morphology looks irregular, peaks are shifted outside of bins
over-concentrated sample fluoresces so much that its signal is refracted into the detection area of another capillary on the camera
can look like a low level profile/contaminant
to resolve - a spatial calibration may need to be performed
76
what are the three conclusions that you can come to about an individual relating to an evidence/unknown profile? briefly describe them
inclusion – an individual cannot be excluded as a potential contributor of DNA obtained from an evidentiary item
exclusion – an individual can be excluded/eliminated as a potential contributor of DNA obtained from an evidentiary item
inconclusive – no conclusion (inclusion/exclusion) can be drawn after comparison to the known
77
what is the difference between a distinguishable mixture vs an indistinguishable mixture?
distinguishable (major/minor) – if there is a clear difference between the amount of DNA from one contributor to the next in a the mixture, then the mixture may be able to be sorted into individual profiles based purely on calculations – 3:1 ratio rule
you have to be able to distinguish a major/minor at at least 18 loci
78
what does insufficient DNA mean?
allele(s) detected above the analytical threshold, but present at less than seven autosomal loci
78
what does non-amplifiable mean?
no alleles were detected above the analytical threshold
79
what does insufficient quant value mean?
greater than or equal to seven autosomal loci detected and total autosomal amplification target of less than 125picogram
80
what three reasons could make a profile limited?
1. less than 18 loci without the possibility of dropout
2. profiles in stochastic range with allelic activity below analytical threshold indicating additional contributor(s) may be present
3. profiles with peak heights at multiple locations indicating additional contributor(s) may be present
81
what three reasons could make a profile complex?
1. the presence of more than 2 contributors
2. the potential biological relationship prohibits the determination of the number of contributors
3. a combination of human and non-specific amplification prohibiting interpretation of the profile
82
when are comparison to reference samples performed?
comparison to reference samples is performed AFTER interpretations of evidence profiles are completed
83
what are the 5 reasons we need/do statistics?
1. FBI QAS requirement
2. NRCII – we have to give some sort of context to the match
3. SWGDAM guidance
4. ISO 17025 & AR 3125
5. with any relevant inclusions, statistical calculations are performed following proper interpretation on evidentiary DNA profiles to provide an assessment of the significance of an inclusion
84
what is probability (P)?
a mathematical relationship between the number of times an event is observed compared to the total number of events possible; the value is always between 0 and 1
85
what is the product rule and why do we use it?
the product rule is used to combine unlinked or independent events using multiplication
for unrelated events (independent), such as locus probabilities of a DNA profile, the probabilities for each locus can be combined using the “product rule”
86
what is likelihood?
conditional probability (likelihood) is the probability an event will occur given knowledge that another event occurred or assuming another event occurred
87
what is a population database?
a population database refers to a collection of observed allele frequencies for a specific group of individuals
88
what stats for STRs do we perform at LSPCL?
random match probability (RMP)
- used with single source samples or a distinguished major contributor in a mixture
- the frequency at which we expect to see the profile we obtained from the evidence within a given population
likelihood ratio (LHR)
- used with two person mixtures that is specific to the observed evidence in the case and comparing specific individuals to that evidence or a distinguished minor in a mixture
- compares two scenarios and presents which of the two is more likely given the evidence
89
what is minimum allele frequency (MAF)? what is the most common equation for the MAF? why is MAF necessary?
MAF refers to the minimum allowable frequency within a population group and is based upon the size of the population sampled
the most common equation – 5/2n
- numerator – minimum number of times that an allele should be seen for a reliable frequency (5)
- denominator – 2 multiplied by the size of the database to account for all observed alleles within a locus
it is used for unobserved alleles and raising frequencies that fall below the MAF
90
what is theta (θ) used for?
theta is a correction factor and is used to address the increase in homozygosity that is observed in our population and to correct for this tendency
by applying theta to homozygous loci, the stat is more conservative (lower)
however, theta is not applied to heterozygous loci as this would artificially inflate the stat
the value we use is 0.01; however, in small/isolated populations “with known inbreeding” the value can be increased to 0.03
91
what is Hardy-Weinberg Equilibrium (HWE)? what are the 5 requirements for HWE?
HWE is the mathematical relationship between genotypes and allele frequencies, but also the idea of a perfectly balanced population where the genetic variation remains constant between generations
p2 + 2pq + q2 = 1
the five requirements are:
1. no mutation
2. random mating
3. no gene flow
4. very large population – no genetic drift
5. no natural selection
92
what is linkage equilibrium?
LE refers to a stable, independent, and random flow of alleles within a population; directly related to HWE and allows for the use of the product rule to combine locus probabilities
93
what is the product rule?
a statistical principle allowing for independent or unlinked events to be combined through multiplication
94
what is a Random Match Probability (RMP)? when do we use this statistic?
the estimated frequency at which a particular STR profile would be expected to occur in a population as determined by the allele frequencies from that population group
it represents the probability of randomly selecting an unrelated individual from the population who could be a potential contributor to an evidentiary profile
probability is a numerical value between 0 and 1
we use this statistic on single source profiles and major contributors in a mixture (if the minor is uninterpretable)
95
what is a Likelihood Ratio (LHR)? when do we use this statistic?
the ratio of two probabilities of the same event under different and mutually exclusive hypotheses
a.k.a. it is the ratio of two conditional probabilities
expressed as a fraction – prosecutor’s hypothesis / defense’s hypothesis
we use LHR for mixtures and minors of mixtures
96
what is the difference between an unrestricted and a restricted LHR? which one do we use on a day to day basis?
unrestricted – all possibilities are considered of the deduced profile regardless of the characteristics of the mixture
- popstats calculates unrestricted LHR!
restricted – means looking at the mixture to attempt to narrow down the possibilities for the subject
- peak heights are utilized to account for all reasonable genotypes that would explain the unknown
97
what is an important limitation of unrestricted LHRs in Popstats?
requires the assumption that all contributors are fully represented at the loci being utilized (no possibility of DO)
98
what is probabilistic genotyping? what are the three interpretation methods?
refers to the use of biological modeling, statistical theory, computer algorithms, and probability distributions to infer genotypes and calculate LHRs for the DNA typing results of forensic samples
three interpretation methods:
1. binary: manual
- treat alleles as present or absent (very much a yes or a no)
2. semi-continuous
3. fully-continuous: TrueAllele
- mathematical modeling of biological parameters
