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Analyst Training > Quantification > Flashcards

Quantification Flashcards

1
Q

what is an autosomal DNA target

A
  • multi-copy
  • target amplicon is 84 base pairs long
  • determines total human DNA in sample
  • smaller target detects degraded DNA
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2
Q

what is autosomal STR testing

A

STRs represent the nuclear DNA within body cells

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3
Q

what is an amplification curve

A

shows accumulation of product as PCR progresses

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4
Q

what is the Cq value and what does it mean

A
  • the Cq vaue is inversely proportional to the amount of starting template
  • a low Cq value indicates less cycles are needed for fluorescence to cross the threshold, indication there is a greater amount of DNA in the sample
  • a high Cq value indicates more cycles are needed for fluorescence to cross the threshold, indicating there is a lower amount of DNA in the sample
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5
Q

what does Cq/t mean

A
  • quantification cycle
  • cycle at which fluorescence exceeds a threshold for baseline noise
  • amplification curve crosses the amplification threshold (Ct)
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6
Q

what is a degradation target and how many base pairs is it

A
  • 294 base pairs long and more susceptible to degradation
  • same locus as the autosomal target
  • autosomal target / degradation target = degradation index
  • degradation index of less than 2 indicates no degradation
  • degradation index of 0 or more than 10 indicates significant degradation
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7
Q

what is emission

A

the instrument optics collect the residual fluorescence emitted from the wells of the reaction plate

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8
Q

what happens during excitation

A

all wells of the reaction plate are illuminated exciting the fluorophores in each well

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9
Q

what happens in the exponential phase

A
  • close to 100% efficiency, amplicon formation is doubling each cycle
  • PCR components are in excess
  • optimal place to measure fluorescence
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10
Q

what is fluorescence detection

A
  • sample wells are illuminated with a bright white light emitting diode (LED)
  • light passes through five excitation filters and the optical adhesive cover before reaching the sample wells
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11
Q

what is an internal PCR control (IPC)

A
  • a novel DNA template included in every Promega quantitative amplification reaction
  • amplification performance of the IPC is used to detect the possible presence of inhibitors in a DNA sample
  • 435 base pairs long, included in the primer mix
  • a shift in IPC or an undetermined IPC means inhibition is present
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12
Q

what is the lag phase

A
  • baseline, before significant product formation
  • background noise
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13
Q

what is the linear phase

A
  • reaction shows efficiency slows to an arithmetic increase
  • PCR components start to fall below critical concentration
  • not useful for comparison purposes
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14
Q

what is the male gDNA standard

A
  • made from purified whole blood from male donors
  • used to generate the dilution series of DNA quant standards
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15
Q

what is mtDNA testing

A
  • testing DNA found in the mitochondria (maternal)
  • used with degraded DNA samples by analyzing shorter segments of DNA
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16
Q

what is a no template control (NTC)

A
  • consists of only quantification amplification reagents without the addition of template DNA
  • used to detect DNA contamination in quantification reagents
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17
Q

what is normalization

A

normalized reporter fluorescence (Rn) = signal of the reporter dye / signal of the passive reference dye

18
Q

what is a passive reference dye

A
  • added to the reaction t capture instrument fluctuations
  • its’ signal is not involved in the PCR process
  • reduces variability
19
Q

what is the plateau phase

A
  • product formation has diminished
  • PCR components have been exhausted and reached the end of their effectiveness
20
Q

explain the polymerase chain reaction (PCR)

A
  • an enzymatic process when a specific region of DNA is replicated over and over again to yield many copies of a particular sequence
  • products are analyzed after cycling is completed
21
Q

what are the components of the PowerQuant kit

A
  • PowerQuant 2X Master Mix
  • PowerQuant 20X Primer/Probe/IPC Mix
  • amplification grade water
  • PowerQuant Male gDNA Standard
  • PowerQuant Dilution Buffer
  • CXR dye (purchased separately)
22
Q

what does the PowerQuant system do

A
  • five-dye, four-target hydrolysis probe-based qPCR multiplex
  • amplifies multicopy targets to quantify the total human and male DNA present in a sample
  • measures an increase in fluorescence over time
23
Q

what is a probe

A
  • DNA sequence that detects and attaches to specific target regions of the template DNA
  • reporter and quencher dye at either end
24
Q

what is a quant negative control

A
  • a negative control
  • no template control (NTC)
25
Q

what does the QuantStudio5 do

A
  • uses fluorescence-based PCR to perform quantitative and qualitative detection of specific targets
  • uses a complimentary metal oxide semiconductor (CMOS) to excite the samples
26
Q

what is quantification

A
  • to determine the quantity and quality of the human DNA available for amplification
  • QAS requirement
27
Q

explain the quantification workflow

A
  • step 1: design plate map, create sample name list, export sample names
  • step 2: plate setup
  • step 3: import sample names, generate powerquant data
  • step 4: export powerquant data
  • step 5: import powerquant data
28
Q

what is the quencher dye

A

a molecule that absorbs the energy of the reporter

29
Q

explain real-time PCR (RT-PCR)

A
  • the most accurate, precise, and efficient method for human DNA quantification
  • detects and measures the exponential accumulation or reduction of light emitted from fluorescent dyes
  • products are monitored while PCR is occurring
30
Q

what is a regression line

A
  • calculated using best fit curve with the quant standard data points
  • Ct/q = m(logQty) + b
31
Q

what is a reporter dye

A

a fluorescent molecule that is used to monitor PCR product accumulation

32
Q

what is the R^2 value

A
  • measure of the closeness of fit between the standard curve regression line and the individual Ct/q data points of the standards
  • 1.0 = perfect fit
33
Q

what does the slope represent on a QS5

A
  • indicates the PCR amplification efficiency for the assay
  • -3.3 = 100% efficiency
  • -3.1 → -3.6 = acceptable range
34
Q

what does the standard curve represent on a QS5

A
  • negative slope
  • places standards with known DNA concentrations on a line of best fit
  • used to assign concentrations to the DNA samples
  • Cq of standards vs. log of concentration
  • uses known concentrations of 50ng, 2ng, 0.08ng, and 0.0032ng
35
Q

what is stop analysis

A
  • if specific criteria is met, samples may be discontinued from the analysis process
  • ex: male/female SA case, but samples show no male DNA
36
Q

what is the taqman probe

A
  • hybridized to complementary targets on the DNA strand
  • cleaves the PCR probe at the reporter end, causing fluorescence
37
Q

what is a treshold

A

fluorescence measurement at which product can be distinguished from the background

38
Q

what is the y-chromosome target and how many base pairs is it

A
  • allows the quantification of a sample’s human male DNA component
  • consists of two multi-copy loci
  • 81 base pairs and 136 base pairs long
39
Q

what does the y-intercept represent on a QS5

A

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

40
Q

what is y-str testing

A

Y-STRs represent the DNA found on the Y chromosome (paternal)

Analyst Training (10 decks)

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