Quantification Flashcards
what is an autosomal DNA target
- multi-copy
- target amplicon is 84 base pairs long
- determines total human DNA in sample
- smaller target detects degraded DNA
what is autosomal STR testing
STRs represent the nuclear DNA within body cells
what is an amplification curve
shows accumulation of product as PCR progresses
what is the Cq value and what does it mean
- 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
what does Cq/t mean
- quantification cycle
- cycle at which fluorescence exceeds a threshold for baseline noise
- amplification curve crosses the amplification threshold (Ct)
what is a degradation target and how many base pairs is it
- 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
what is emission
the instrument optics collect the residual fluorescence emitted from the wells of the reaction plate
what happens during excitation
all wells of the reaction plate are illuminated exciting the fluorophores in each well
what happens in the exponential phase
- close to 100% efficiency, amplicon formation is doubling each cycle
- PCR components are in excess
- optimal place to measure fluorescence
what is fluorescence detection
- 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
what is an internal PCR control (IPC)
- 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
what is the lag phase
- baseline, before significant product formation
- background noise
what is the linear phase
- reaction shows efficiency slows to an arithmetic increase
- PCR components start to fall below critical concentration
- not useful for comparison purposes
what is the male gDNA standard
- made from purified whole blood from male donors
- used to generate the dilution series of DNA quant standards
what is mtDNA testing
- testing DNA found in the mitochondria (maternal)
- used with degraded DNA samples by analyzing shorter segments of DNA
what is a no template control (NTC)
- consists of only quantification amplification reagents without the addition of template DNA
- used to detect DNA contamination in quantification reagents
what is normalization
normalized reporter fluorescence (Rn) = signal of the reporter dye / signal of the passive reference dye
what is a passive reference dye
- added to the reaction t capture instrument fluctuations
- its’ signal is not involved in the PCR process
- reduces variability
what is the plateau phase
- product formation has diminished
- PCR components have been exhausted and reached the end of their effectiveness
explain the polymerase chain reaction (PCR)
- 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
what are the components of the PowerQuant kit
- PowerQuant 2X Master Mix
- PowerQuant 20X Primer/Probe/IPC Mix
- amplification grade water
- PowerQuant Male gDNA Standard
- PowerQuant Dilution Buffer
- CXR dye (purchased separately)
what does the PowerQuant system do
- 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
what is a probe
- DNA sequence that detects and attaches to specific target regions of the template DNA
- reporter and quencher dye at either end
what is a quant negative control
- a negative control
- no template control (NTC)
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
what is quantification
- to determine the quantity and quality of the human DNA available for amplification
- QAS requirement
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
what is the quencher dye
a molecule that absorbs the energy of the reporter
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
what is a regression line
- calculated using best fit curve with the quant standard data points
- Ct/q = m(logQty) + b
what is a reporter dye
a fluorescent molecule that is used to monitor PCR product accumulation
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
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
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
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
what is the taqman probe
- hybridized to complementary targets on the DNA strand
- cleaves the PCR probe at the reporter end, causing fluorescence
what is a treshold
fluorescence measurement at which product can be distinguished from the background
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
what does the y-intercept represent on a QS5
indicates the expected Ct/q value for a sample with a quantity of 1ng/ul
what is y-str testing
Y-STRs represent the DNA found on the Y chromosome (paternal)
