Chapter 6 - DNA Quantitation
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37 Terms
Purpose/Why is determining DNA amount essential
PCR amplification is dependent on the quantity of DNA in the sample
DNA quantitation Techniques
Yield gel, slot blot, picogreen, real time quantitative PCR
Yield Gel: information obtained
Amount of DNA
Quality of DNA
Yield Gel: limitations
Cannot differentiate between human & non-human DNA
Not very sensitive
Cannot be used to quantify samples extracted using methods that result in single-stranded DNA
Yield gel procedures
Load samples with tracking dye onto 1% agarose gel
DNA runs toward cathode
Soak in ethidium bromide (EB)
Expose to UV light to excite EB
Amount of fluorescence is proportional to the total amount of DNA
Slot blot: Information obtained
Amount of DNA
NO information on quality
Slot blot Advantages
Higher sensitivity than yield gel
Probe designed to only recognize primate DNA
slot blot Disadvantages
No information on DNA quality
Test takes longer
Requires more equipment
Not very sensitive to degraded DNA
slot blot procedures
Prepare samples and standards
Apply both to the membrane
Place plastic tray over membrane
Attach vacuum to attach samples to membrane
Hybridize probe to membrane
Wash to remove unattached probe
Detect probe with horseradish peroxidase
Compare color of standards to color of unknown
Estimate concentration
slot blot How to read/interpret results
just compare to standards
pico green info obtained
Enhanced fluorescence when bound to DNA
type of dye used in pico green
PicoGreen is a fluorescent intercalating dye
pico green procedure
d 5 μL of sample to 195 μL of solution containing dye in 96-well plate
Examine plate with fluorimeter in under 30 minutes
Compare unknown fluorescence to standard curve
Real-Time Quantitative PCR
determines the amount of amplifiable DNA
What is RT-PCR
real time polymerase chain reaction
Monitors the fluorescence emitted during PCR as an indicator of the product created after each cycle
qPCR
quantitative polymerase chain reaction
PCR is performed in a specialized thermal cycler that can measure the amount of product after all cycles are complete
o TaqMan
Labeled with two fluorescent dyes that emit at different wavelengths
Taxman how does it work
Probe sequence hybridized to a specific DNA sequence between forward & reverse primers
Reporter dye is at 5’ end, quencher dye is at 3’ end
o Why Does RT-PCR work
Based on the detection & quantitation of a fluorescent signal
Signal increases in direct proportion to the amount of PCR product in the reaction
The rate at which PCR product accumulates is a function of the initial template in the reaction
Increased template = increased amplicon production
In real time, qPCR amplification curves are constructed for each PCR reaction by detection & quantification of a fluorescent reporter
The form of the curve is a function of the amount of starting template
o Quantifiler How does it work
DNA quantitation is important to determine how much human DNA (as opposed to bacterial DNA) is present in a sample
o Quantifiler gene target
Human telomerase reverse transcriptase gene (hTERT)
Cycle threshold (CT)
o The threshold setting defines the level of detectable fluorescence
what does Cycle threshold (CT) depend on
Starting template copy number
Efficiency of DNA amplification by the PCR system
Total human vs. male specific
RT-PCR output and the 4 phases of amplification
o Lag (doubling, but not detected)
o Exponential (doubling)
o Linear (less than doubling)
o Plateau (little change)
Identify the different areas of the graph
o The exponential phase is where we make our qPCR measurements
Male:Female DNA ratio
In DNA mixtures of male and female individuals, it may be useful to calculate the ratio of total autosomal DNA to the male-specific Y-chromosome DNA
The quantity of human DNA in this calculation is based on the quantity value for the small autosomal target.
For example, assuming:
o Male DNA concentration = 2 ng/μL
o Human DNA concentration = 8 ng/μL
Degradation Index
is automatically calculated by the HID Real-Time PCR Software using the following formula:
Small autosomal target [DNA] (ng/µL)
Large autosomal target [DNA] (ng/µL)
is a good gauge of whether profile results will indicate degradation of sample
IPC CT Flag/analysis
is triggered for an unknown sample that has an IPC CT of:
o Undetermined
o Greater than the average of the IPC CT values for all the standards plus the threshold you set in the software HID Settings
when this is triggered it indicates that inhibitors are present
Quality Index
can help you determine next steps, including:
o Proceed directly to an STR analysis of the sample
o Dilute the sample before adding to the STR reaction
o Perform additional cleanup of the sample to remove potential inhibitors and requantify the sample if necessary
o Use one of the next generation STR kits for improved performance with inhibited samples
o Use an STR assay that includes a high number of miniSTR loci, such as the GlobalFiler™ and MiniFiler™ PCR Amplification Kits for increased data recovery from degraded samples
o Importance of the calibrant
The Calibrant is usually a pristine well-characterized DNA sample
o Not extracted
o Not subjected to the same environment as your unknown(s)
o Will not contain inhibitors, Ca++ etc
o May be from a cell line or mixed source sample
o May exhibit lot-to-lot variation (must be monitored)
What is happening during rtqPcr
In RT-qPCR the products are monitored as the PCR is occurring (dynamic)
Once per thermal cycle
Fluorescence is measured
Kinetics of the system
What is happening during qpcr
In qPCR the products are analyzed after the cycling is completed (static)
gel, CE, UV, fluorimeter
End point assay
qPCR Advantages
o No post PCR manipulation (reduced contamination issues)
o High sensitivity (down to a single copy number ?)
o Large dynamic range: ~30 pg to ~30 ng
o Assays are target specific (autosomal, mito, Y) and can be multiplexed – to a degree…
qPCR Disadvantages
o qPCR is subject to inhibition
internal PCR controls (IPC) can help
o qPCR quantitation precision suffers at low copy numbers (below 30 pg by a factor of 2)
o When working below 100 pg qPCR is still subject to variability and uncertainty
Small changes in CT means large change in quantity
PCR Disadvantages:
o Inhibitors can affect results
o May not be accurate for LCN & degraded DNA
o Assay assumes standards amplify the same as the unknowns
PCR Advantages:
o Very sensitive (down to 5-10 cells)
o Can target specific human DNA sequences
o Simple test protocol, kits available
o Can do multiplex reactions
o Automated, computer software