Quantification Flashcards
(True or False) The human male (Y) target is single copy to reduce variability between individuals and to correlate with STR amplicons.
False; the human male target is multicopy
When using the QuantStudio instrument, light passes through five ______ and the ______ adhesive cover before reaching the samples in the ______.
Excitation Filters
Optical
Wells
What is the name of the chemistry that is used in many quant kits?
TaqMan Chemistry
What organism was Taq DNA polymerase originally isolated from?
Thermus Aquaticus
What is the amplicon length of the human (autosomal) target for the PowerQuant System?
84 bases
What is the purpose of a passive reference dye in qPCR?
Normalization
What is the amplicon length(s) of the human male (Y) target for the PowerQuant System?
81 bases and 136 bases
What qPCR phase shows that no more product is being produced?
Plateau
What volume of sample, control, and DNA standards are added to a PowerQuant reaction?
2 microliters
What is the phase of qPCR that shows a slowing of the amplification efficiency?
Linear
What is the reaction volume of a PowerQuant reaction?
20 microliters
What is the point in which fluorescence passes the baseline noise in qPCR?
Cycle Threshold or Quantification Cycle
Why is quantification used in forensic DNA testing?
To determine the quantity and quality of human DNA
A sample with inhibition would have a/an ______ IPC CT value
Elevated
What is the measure of the closeness of fit between the standard curve regression line and the individual CT data points?
R squared value
(True or False) The TaqMan chemistry uses probes with a reporter dye and a quencher that anneal within a target sequence outside forward and reverse primers.
False; between forward and reverse primers
The CT value is ______ proportional to the ______ of the DNA sample.
Inversely
Concentration
What is the passive reference dye for PowerQuant?
CXR
What is a feature of modern qPCR chemistries that can show inhibition within a sample?
Internal PCR Control (IPC)
What must be prepared and added to a qPCR run in order to determine the concentration of a DNA sample?
DNA Standard Dilution Series
Which qPCR phase is related to the doubling of amplicons in each cycle at 100% efficiency?
Exponential
What slope indicates 100% PCR amplification efficiency?
-3.3
What is the stock concentration of the PowerQuant standard?
50 nanogram/microliter
(True or False) The human (autosomal) target is multicopy to reduce variability between individuals and to correlate with STR amplicons.
True
How many standard dilution points should be created when using the PowerQuant System at LSPCL?
4
What does the slope of the standard curve indicate?
PCR efficiency
What does the y-intercept of the standard curve indicate?
It is the expected ct/cq value for a sample at 1ng
Does the PowerQuant System measure an increase or decrease in fluorescence?
Increase in fluorescence
