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
2
Q
what is autosomal STR testing
A
STRs represent the nuclear DNA within body cells
3
Q
what is an amplification curve
A
shows accumulation of product as PCR progresses
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
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)
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
7
Q
what is emission
A
the instrument optics collect the residual fluorescence emitted from the wells of the reaction plate
8
Q
what happens during excitation
A
all wells of the reaction plate are illuminated exciting the fluorophores in each well
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
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
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
12
Q
what is the lag phase
A
- baseline, before significant product formation
- background noise
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
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
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
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
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
what does the QuantStudio5 do
- 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
what is quantification
- to determine the quantity and quality of the human DNA available for amplification
- QAS requirement
27
explain the quantification workflow
- 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
what is the quencher dye
a molecule that absorbs the energy of the reporter
29
explain real-time PCR (RT-PCR)
- 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
what is a regression line
- calculated using best fit curve with the quant standard data points
- Ct/q = m(logQty) + b
31
what is a reporter dye
a fluorescent molecule that is used to monitor PCR product accumulation
32
what is the R^2 value
- 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
what does the slope represent on a QS5
- indicates the PCR amplification efficiency for the assay
- -3.3 = 100% efficiency
- -3.1 → -3.6 = acceptable range
34
what does the standard curve represent on a QS5
- 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
what is stop analysis
- 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
what is the taqman probe
- hybridized to complementary targets on the DNA strand
- cleaves the PCR probe at the reporter end, causing fluorescence
37
what is a treshold
fluorescence measurement at which product can be distinguished from the background
38
what is the y-chromosome target and how many base pairs is it
- 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
what does the y-intercept represent on a QS5
indicates the expected Ct/q value for a sample with a quantity of 1ng/ul
40
what is y-str testing
Y-STRs represent the DNA found on the Y chromosome (paternal)
