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analyst training > Technical Oral Exam > Flashcards

Technical Oral Exam Flashcards

1
Q

what is an STR?

A

short tandem repeat - short DNA sequences that repeat several times in a row
variations in these repeat units at a locus are called alleles
these highly repetitive sequences are the preferred genetic markers & are amplified using PCR

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2
Q

describe the concept of probability (P)

A

mathematical relationship between the number of times an event is observed compared to the number of events possible (value between 0 and 1)
statistics in forensic DNA utilize allele frequencies from a population database to approximate the probability of observing a profile in the population
for example: rolling a 2 on a 6-sided die

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2
Q

describe TaqMan chemistry

A

utilization of Taq polymerase & TaqMan probe & primers to amplify DNA during quantification
TaqMan probe hybridizes to complementary targets on DNA strand
Taq polymerase cleaves the dual-labeled probe during hybridization of the complementary target sequence
the resulting fluorescence signal permits quantitative measurements of the accumulation of product
denaturation -> annealing -> extension

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2
Q

what is an allele?

A

variation of the number of repeat units within a targeted STR marker
repeat patterns of STRs are located in areas called loci (locus) - variants at a locus are called alleles

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2
Q

what are primers?

A

small fragments of chemically synthesized oligonucleotides that target & flank desired DNA segments to be amplified
typically 15-25 bases long
added in high concentrations compared to template DNA to drive amplification process forward
forward and reverse primers

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3
Q

what is the most common method of dye labeling for forensic STR DNA analysis?

A

dye-labeled primers
dye attached to 5’ end of the forward primer

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4
Q

what is a likelihood ratio (LR)?

A

the ratio of two probabilities of the same event under different & mutually exclusive hypotheses
statistic that is specific to the observed evidence in the case & comparing specific individuals to that evidence
ratio of two conditional probabilities
used for mixtures & minors of mixtures

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5
Q

what does mutually exclusive mean in regards to LR?

A

if hypothesis 1 is true, then hypothesis 2 canNOT also be true

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6
Q

what is the Ct/Cq value?

A

cycle threshold/quantification cycle
cycle at which fluorescence exceeds a threshold for baseline noise
inversely proportional to the amount of starting template DNA
low Cq value = less cycles needed to cross threshold = more DNA
high Cq value = more cycles needed to cross threshold = less DNA

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7
Q

what is DNA?

A

deoxyribonucleic acid
you get half from your mom, half from your dad
the building blocks of what makes each ofus who we are
a self-replicating material that is present in nearly all living organisms as the main constituent of chromosomes
it is the carrier of genetic information

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8
Q

what are probes?

A

short DNA sequence that detects & attaches to specific target regions of template DNA
contains a reporter dye (fluorophore) and quencher (molecule that quenches the fluorophore)
when the quencher is in close proximity to reporter, reporter fluorophore will not fluoresce
once Taq polymerase degrades the probe, the reporter and quencher disassociate and the reporter fluoresces
fluorescence from reporter is proportional to the amount of amplified product (DNA)

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9
Q

what is a random match probability (RMP)?

A

estimated frequency at which a STR profile would be expected to occur in a population as determined by the allele frequencies from that population group
we report the RMP frequency, not the RMP itself (1/RMP)
used on single source profiles and major contributor of mixture profiles

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10
Q

what is Taq polymerase?

A

originated from Thermus aquaticus
adds complementary dNTPs (of template strand) to new DNA fragment one at a time

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11
Q

why do we report the RMP frequency and not the RMP itself?

A
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12
Q

describe our PowerQuant kit

A

five-dye, four-target hydrolysis probe-based qPCR multiplex that amplifies multicopy targets to quantify the total human & human male DNA present in a sample
four targets: autosomal DNA, Y (male) DNA, degradation, inhibition
measures an increase in fluorescence overtime

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13
Q

why is Taq polymerase used over other DNA polymerases?

A

thermostability
prevents non-specific PCR products & primer dimers from forming before activation and allows for room temperature set-up before amplification

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14
Q

describe our Fusion 6C amplification kit

A

six-dye multiplex system
27 total loci detection (23 autosomal, 4 sex determining)

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15
Q

what are the pros of using the Fusion 6C kit?

A

high sensitivity & tolerance to inhibitors
shorter amp time due to optimized Taq polymerase (short hot start & fast DNA synthesis)
improved primers enhance signal-to-noise ratio & simplify interpretation
power of discrimination is increased

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16
Q

what does multiplex mean in regards to the F6C kit?

A
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17
Q

what is the instrument we use in amplification and describe the cycles

A

thermalcycler
incubation - 96C for 1 min
*denature - 96C for 5 sec
*anneal/extension - 60C for 1 min
*29 cycles
final extenstion - 60C for 10 min
4C soak
total time - ~60 min

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18
Q

what is the function of magnesium chloride in a PCR rxn?

A

co-factor required by DNA polymerase to function
aids in primer annealing
increasing concentration increases yield, but decreases specificity & fidelity
presence of EDTA & other chelators might disturb magnesium optimum

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18
Q

what is the function of potassium chloride in a PCR rxn?

A

neutralizes charge present on DNA backbone
facilitates primer annealing to template DNA
reduces repulsion between negatively charged DNA strands (primers & templates) thereby stabilizing primer template binding

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19
Q

what may the concentration magnesium chloride in a PCR rxn affect?

A

primer annealing
product specificity
formation of primer-dimer artifacts
enzyme activity & fidelity
strand disassociation temperatures of template &/or PCR product

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20
Q

describe the inner capillary environment

A

the capillary itself is glass and is silica-infused and is negatively charged (from the silica)
anode buffer’s positive ions line the wall & create the electroosmotic flow
linear, flexible polymer chains act as a sieve/obstacles for the DNA - polymer reduces backward flow so DNA can still move toward positive charge (speed of DNA depends of size alone, not charge-to-mass ratio)
DNA moves through the capillary from the cathode (-) to the anode (+)

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21
Q

describe the electrophoretic flow vs. the electroosmotic flow

A

EPF - DNA (-) moves away from the cathode (-) towards the anode (+)
EOF - opposite of the EPF, the anode (+) buffer flows towards the cathode (-)

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22
Q

what is the function of bovine serum albumin (BSA) in a PCR rxn?

A

protein added to help stabilize polymerase
reduces/prevents PCR inhibition

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23
Q

what are the 8 PCR components needed for amplification?

A
  1. DNA (template DNA)
  2. primers
  3. deoxynucleoside triphosphates (dNTPs)
  4. DNA polymerase
  5. bovine serum albumin (BSA)
  6. magnesium chloride
  7. potassium chloride
  8. tris-HCl buffer
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24
Q

what are the lengths of the 4 targets in quantification?

A

autosomal DNA - 84 bp
Y (male) DNA - 81 bp & 136 bp
degradation - 294 bp
inhibition - 435 bp

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25
Q

describe the purpose of the passive reference dye

A

CXR dye
present in all wells of rxn plate at same concentration
purpose - normalization; differentiate background noise of fluctuations of instrument from the fluorescence levels of DNA in sample

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26
Q

describe the quantification standard curve

A

graph of the Cq values of the standards vs. the Log of the concentration of the standards
slope (regression line) indicates PCR efficiency
regression line is calculated using best fit curve with quant standard data points
utilizes standard dilution series
used to determine the concentration of unknown DNA samples (software calculates DNA concentration based on Ct/q value of sample & comparing it to the standard curves Ct/q’s

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27
Q

what is the slope/regression line formula of the standard curve?

A

Ct/q = m(logQty)+b

Qty = starting amount of DNA

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28
Q

how many standards do we utilize and what are their concentrations?

A

4

  1. 50 ng/ul
  2. 2 ng/ul
  3. 0.08 ng/ul
  4. 0.0032 ng/ul
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29
Q

what does the y-intercept indicate on the quantification standard curve?

A

the expected Ct/q value for a sample with a quantity of 1 ng

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29
Q

what does the R2 value indicate on the quantification standard curve?

A

measure of closeness of fit between the standard curve regression line & individual Ct/q data points of standards

1 perfect fit / >/= 0.99 passing R2 value

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29
Q

what does the slope indicate on the quantification standard curve?

A

PCR efficiency

-3.3 100% efficient / -3.1 - -3.6 passing slope

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30
Q

describe the standards in quantification

A

made from purified whole blood from multiple male donors
expires after 7 days
critical for accurate analysis of run data
stock concentration is 50ng/ul

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31
Q

what are the 3 requirements for STR typing in capillary electrophoresis?

A
  1. spatial resolution
  2. spectral resolution
  3. DNA sizing precision
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32
Q

why is spatial resolution required for CE?

A

it determines the relationship between the signal emitted from each capillary & where it’s detected on the CCD camera - it maps the position of each capillary to a region on the camera
necessary to separate DNA fragments that may differ in size by a single nucleotide

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33
Q

why is spectral resolution required for CE?

A

helps separate out spectral dye overlap
necessary to separate fluorescent dye colors from one another so that dye labeled PCR products from different loci can be resolved

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34
Q

why is DNA sizing precision required for CE?

A

must be consistent from run to run enough so that samples can be related to allelic ladders & yield reproducible results

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35
Q

what is ISO?

A

International Organization of Standards
ISO17025 - required for testing & calibration laboratories

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36
Q

what is AR?

A

Amended Reports
AR3125 - required for forensic testing & calibration laboratories

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37
Q

what is QAS?

A

Quality Assurance Standards
FBI QAS - set forth by FBI for DNA testing laboratories

38
Q

how many active sets of QAS are there?

A

2
one for forensics and one from databasing

39
Q

how many main standards are in each QAS document?

A

17 main standards

40
Q

what is SWGDAM?

A

Scientific Working Group on DNA Analysis Methods
publishes guidelines or recommendations for best practices in forensic DNA laboratories - how to best implement ISO & QAS standards into your lab

41
Q

describe differential extractions

A

isolation of two cell types - epithelial cells & sperm cells to extract DNA
accomplished by selectively lysing non-sperm cells & removing the non-sperm DNA prior to lysing sperm cells
sperm have stronger cell membranes than epithelial cells & in turn are more difficult to lyse

42
Q

what are 4 things that may impact results of a differential extraction?

A
  1. there may be more epithelial cells than sperm cells
  2. incomplete initial digestion
  3. inadequate washing of sperm
  4. carryover of F1 into F2
43
Q

what is DTT?

A

dithiothreitol
reduces disulfide bonds of proteins in cell membranes allowing release of the DNA
especially crucial for digesting hair and sperm cells

44
Q

what is pK?

A

proteinase K
serine protease - cleaves peptide bonds in proteins imbedded in cell membranes
enzyme used to digest proteins (histones) to expose the DNA
inactivates nucleases that break down DNA

45
Q

what is ATL?

A

tissue lysis buffer
lysis of cell membranes to release & expose DNA

46
Q

what is cRNA?

A

carrier RNA
enhances binding of DNA to surface of magnetic particles, especially hen low amounts of DNA is expected
drives binding process
added to increase DNA yield in low concentration samples

47
Q

what is G2?

A

tissue lysis buffer
lysis of cell membranes to release & expose DNA
less harsh than ATL!
used in differential extractions to selectively lyse epithelial cells before sperm cells

48
Q

what is MTL?

A

lysis buffer with chaotropic salts used together with EZ2 for automated purification
large volume samples

49
Q

describe the Local Southern Method

A

GMID-X utilizes our ILS (WEN) to size unknown DNA fragments
it takes 4 fragments closest to the unknown fragment & creates two size curves
the first size curve utilizes two peaks before & one peak after
the second size curve utilizes one peak before and two peaks after
it takes the average of these two size curves & gives you the base pair size of our unknown DNA fragment

50
Q

x-axis vs. y-axis on an electropherogram

A

x-axis - plots the base pair size of DNA fragments
y-axis - plots the rfu value (concentration of DNA present)

51
Q

what is an electropherogram?

A

GMID-X utilizes the raw data from our CE runs to plot it onto an electropherogram so we can easily read it. it plots the size of the DNA fragments (by number of base pairs; x-axis) and the concentration of the DNA (rfu value; y-axis)

52
Q

what is a null allele?

A

an allele present in the sample, yet it is not amplified
often caused by primer binding sit mutations (if the primer doesn’t bind, amplification does not occur)

53
Q

describe the analytical threshold

A

defines the height requirement at and above which detected peaks can be reliably distinguished from background noise (100rfu)

54
Q

describe the saturation threshold

A

only used when pull-up is seen at 100rfu
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 AT causing artifacts to be labeled as true peaks (300rfu)

55
Q

describe the stochastic threshold

A

peak height or signal magnitude below which it is reasonable to assume that allelic DO of a sister allele in a heterozygous pair may have occurred (350rfu)
ST was determined during validation studies using sensitivity data (known profiles at decreasing concentrations)

56
Q

what are the 7 steps to interpret data

A
  1. determine the minimum number of contributors
  2. maj/min or indistinguishable?
  3. interpretable or uninterpretable?
  4. designate genotypes
  5. utilize available assumptions for further resolution of genotypes if appropriate
  6. compare evidence to applicable references
  7. perform statistical evaluation if applicable
57
Q

describe Hardy-Weinberg Equilibrium

A

p2 + 2pq + q2 = 1

mathematical relationship between allele frequencies and genotype frequencies
principle of a perfectly balanced population where the genetic variation remains constant between generations in the absence of disturbing factors
HWE is dependent on Mendel’s law of segregation & independent assortment of genes during sex cell formation

58
Q

what is the function of tris-HCl buffer in a PCR rxn?

A

provides a stable pH environment (pH can alter fidelity of a dNTP insertion)
pH between 8.3-8.8 is used (@ 20C) - pH decreases to 6.9 when heated to 96C to activate Taq

59
Q

describe Linkage Equilibrium

A

refers to a stable, independent, and random flow of alleles within a population
directly related to the ability to achieve or approach HWE & uses the product rule to combine locus probabilities
LE is also dependent on Mendel’s law of segregation & independent assortment of genes during sex cell formation

60
Q

describe theta correction

A

correction factor used to address the issue of increased homozygosity from a population substructure
accounts for the difference between population genotype frequencies & true HWE
0.1 or 0.3

61
Q

what could uncorrected homozygosity cause? uncorrected heterozygosity?

A

homo - could be unjustly prejudicial towards a defendant, especially in a smaller population, by raising a statistic
hetero - could benefit a defendant by lowering a statistic

62
Q

why is it important to know if the considered population is relatively small or large, or a possible population substructure based on ethnicities or beliefs?

A

setting theta too high = overcorrect & possibly reduce the statistic inappropriately (too much)
setting theta too low = undercorrect & possibly reduce the statistic insufficiently (too little)

63
Q

describe why theta is only applied to homozygous loci and not heterozygous loci too

A

overall, the rarity of the genotype is REDUCED with increasing theta values in homozygous loci (more conservative). for heterozygous loci, the rarity is INCREASED with increasing theta values (less conservative)
thus, theta is only applied to homozygous loci & not heterozygous loci in order to calculate a more conservative estimate

64
Q

what are the correct HWE formulas used in the HW equation for homozygous loci and heterozygous loci?

A

heterozygous: f = 2pq
homozygous: f = p2 +p(1-p)theta value
- instead of p2

65
Q

describe the product rule

A

statistical principle allowing unlinked or independent events to be combined 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 (“and” means multiplication)
if two events are independent (A & B have no effect on each other), we can calculate the probability of A AND B by multiplication

66
Q

describe minimum allele frequency (MAF)

A

minimum allowable frequency within a population group & is based on the size of the population sample
used for unobserved alleles & raising the frequencies that fall below the MAF
if the allele is observed fewer than 5 times, Popstats will assign a MAF of 5/2n, which assumes a minimum of 5 observations of that allele in the sample size ‘n’

67
Q

describe the most common MAF equation

A

5 / 2n

the numerator (5) is the minimum number of times that an allele should be seen for a reliable frequency
the denominator (2n) is 2 multiplied by size of the database to account for all observed alleles within a locus

68
Q

why is applying a MAF necessary?

A

if an allele is rare (observed few or 0 times in the sample database), we must provide a conservative estimate for use in statistics - so we don’t skew the statistic too much ( we need to be conservative)

69
Q

describe a restricted likelihood ratio

A

this 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
Popstats is not sophisticated enough to do this

70
Q

describe an unrestricted likelihood ratio

A

this means that all possibilities are considered of the deduced profile regardless of the characteristics of the mixture
using the unrestricted LR would account for all possible genotypes that could explain the unknown
this is what Popstats calculates

71
Q

what is the defendant’s fallacy (numerical conversion fallacy) on an RMP?

A

suppose you have an RMP of 1 in 1 million. the defense may argue, “Louisiana has a population of almost 5 million people. this means there are 4 other people i La alone that could have left that profile”

72
Q

what is an important limitation of an unrestricted LR

A

it requires the assumption that all contributors are fully represented at the loci being utilized (no possibility of DO)

73
Q

what is the prosecutor’s fallacy on an RMP?

A

the defendant’s fallacy can be manifested from the prosecutor as well: suppose you have an RMP of 1 in 1 quadrillion. the prosecutor may ask, “the world population is approximately 7 billion people. how many worlds would you need to see this profile again?”

74
Q

how do you correct the fallacies of an RMP?

A

the RMP refers to an outcome probability over one trial, not how many times that outcome is expected over several trials
so, given that the profile has been observed once already, it describes the probability of observing this profile again if a random person is selected from the population. each time a person is selected, the probability is 1 in value

75
Q

what are the 5 requirements for a population to be in HWE?

A
  1. random mating
  2. large population
  3. no gene flow
  4. no mutations
  5. no natural selection
76
Q

why is random mating a requirement/necessary for HWE?

A

prevents inbreeding &/or the occurrence of a population substructure (amish people)

77
Q

why is a large population a requirement/necessary for HWE?

A

ensures allele frequencies are not changed through genetic drift

78
Q

why is no gene flow a requirement/necessary for HWE?

A

this would increase variability in the gene pool

79
Q

why is no mutations a requirement/necessary for HWE?

A

to avoid introducing new alleles into a population

80
Q

why is no natural selection a requirement/necessary for HWE?

A

stronger genes may be ‘chosen’ over inferior genes which would cause allele frequencies to change if alleles were being favored or lost

81
Q

what is the difference between genetic drift and gene flow?

A

GD - small population (vertical, inbreeding)
GF - large population to large population (horizontal)

82
Q

we are not in HWE (HWD). how can we still use this equation for our statistics?

A

scientists performed an Exact Test (simulation that compared a population in perfect HWE to our population that’s in HWD) & found:
1. no significant deviation from HWE
2. loci are sufficiently discriminatory
3. there are significant differences in allele frequencies among different population groups
4. loci are sufficiently independent (no evidence of association), so the product rule is valid

83
Q

how do we compensate the fact that we are in HWD in regards to our work?

A

genetic markers are chosen that are less affected by disruption
alleles chosen are from loci for noncoding regions of DNA (not expressed as phenotypes, reducing genetic divergence, & loci chosen are typically spaced far apart or on different chromosome (ensuring independence))

84
Q

describe CODIS

A

Combined DNA Index System
FBI’s program of support for criminal justice DNA databases as well as the software used to run these databases
purpose: to generate investigative leads for unsolved cases, to identify UHR, & help solve missing persons cases
3 hierarchal levels: NDIS (national), SDIS (state), LDIS (local)

85
Q

what are the 6 CODIS specimen categories?

A
  1. forensic unknown
  2. forensic partial
  3. forensic mixture
  4. forensic targeted
  5. SDIS forensic
  6. SDIS mixture
86
Q

what makes a profile a forensic unknown?

A

complete, single source or deduced single source profile

87
Q

what makes a profile a forensic partial?

A

single source or deduced single source incomplete profile
MME value of >1E+007

88
Q

what makes a profile a forensic mixture?

A

mixture with >3 alleles at two or more loci & has an MME value of >1E+007

89
Q

what makes a profile a forensic targeted?

A

MME value of <1E+007
MRE value of >1E+007

90
Q

what makes a profile a SDIS forensic?

A

single source
MME value of >1E+005
MRE value of <1E+007

91
Q

what makes a profile a SDIS mixture?

A

mixture
MME value of >1E+005
MRE value of <1E+007
^ same as SDIS forensic

92
Q

what is a MME value?

A

moderate match estimation
calculates estimated match probability value for all specimens in a selected specimen category; not configurable
calculated evaluating 13 CODIS core loci at moderate stringency

93
Q

what is a MRE value?

A

match rarity estimation
calculates estimated rarity value for all specimens in a selected specimen category, has many configurations
locus stringency can be changed
can be calculated for the 20 expanded loci

94
Q

MME & MRE are both inversions of ______ ______ ______, but differ based upon ________.
elaborate on this idea

A

match rarity estimation ; stringency

MME is match rarity estimate when the 13 original core loci are used & all stringencies are set to moderate

MRE is match rarity estimate when you calculate stringency by locus; hetero loci are searched at HIGH stringency; homo loci are searched at MODERATE stringency

95
Q

describe Mendel’s law of segragation

A

during gamete formation, the two alleles at a gene locus segregate from each other; each gamete has an equal probability of containing either allele
during meiosis when sex cells divide

96
Q

describe DISC

A

DNA Index of Special Concern
virtual index of forensic unknowns in CODIS that meet certain eligibility requirements
created to facilitate searching of Arrestee Rapid DNA profiles enrolled in CODIS from booking stations

97
Q

what are the 5 requirements for a profile to be entered onto the DISC?

A
  1. homicide, SA, terrorism, kidnapping
  2. forensic unknown
  3. must be complete at 13 original CODIS core loci
  4. source ID = NO
  5. must have required casework metadata (investigating agency info, offense description, etc.)
98
Q

describe stringency (CODIS)

A

used as a filter to report only locus matches of equal or higher quality

locus stringency - locus to locus
high - exact match
moderate - ‘fits in the bucket’
low - one allele matches, one doesn’t

match stringency - profile as a whole
high - all loci match
moderate - at least one locus is at moderate; none at low
low - at least one locus is at low

99
Q

describe primer-dimers

A

when primers interact with each other & bind to each other
polymerase can extend the annealed primers to produce primer-dimers which compete for reagents in a PCR reaction

100
Q

what are the 4 phases of amplification? describe each phase

A
  1. lag phase - baseline; before significant product formation
  2. exponential phase - close to 100% efficiency, amplicon formation is doubling each cycle
  3. linear phase - rxn efficiency slows to an arithmetic increase; PCR components fall below critical concentration
  4. plateau phase - product formation has diminished; PCR components have been exhausted
101
Q

describe the process of extraction on the EZ2

A

lyse - break open the cells to expose the DNA
bind - chaotropic salts introduced via reagents reduce the pH and causes the DNA to bind to the silica beads
wash - a system of washes get rid of all of the other components and debris other than the DNA
elute - the chaotropic salts are washed away and the pH is raised again causing the DNA to elute into the elution tube

analyst training (4 decks)

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