quantitation Flashcards
dna quantitation
essential for PCR based DNA analysis
PCR based testing is very sensitive
has a narrow conc rane
too much DNA template results in DNA artifacts (extra peaks) that interfere with dna interpretation
too little template may result in partial dna profiles or failure to obtain any profile
amount of dna in the sample will determine how much of the sample will be amplified for str analysis
dna quantitation methods should be human specific for per based analysis
because primers are human specific
forensic samples may include dna from bacteria or other sources
dna quantitation methods which can measure the quality and quantity of human dna are desireabe
forensic methods include: slot blot
interchelating dye
pcr-qpcr and end point
slot blot assay
can be used to detect human genomic dna in a sample
dna is denatured (ssDNA) and small volume is spotted (blot) on to a nitrocellulose membrane or nylon membrane and immobilized
targeted sequence is revealed by hybridization with a labeled 40 nucleotide probe which is added and allowed to anneal
complimentary to a primate specific α-satellite DNA sequence D17Z1 locus
3 detecting labeling schemes have been used
radioisotopes visualized using x-ray film (hazardous)
alkaline phosphatase labeled or biotinylated probes
labels or probes are coupled with chemiluminescent to colorimetric detection
signal intensity is proportional to dna conc
quantitative measurements of unknown samples are taken by comparing the intensity of the signal to a set of standards with known conc
typically can quantify in the range of 150pg-10ng
cross reacts with primate DNA, usually not a problem
fluorescent interchelating dye assay
quantify small quantities of DNA 250pg
uses a fluorescent intercalating dye
inserts itself between bases
upon excitation, releases a signal
needs dsDNA
can’t do in conjunction with chelex (produce ssDNA)
not human specific
was useful for known reference samples
adapted for automation high throughput measured using a standard spectrofluorometer with excitation and emission eavelengths
a standard curve is first created using samples with known DNA conc (standards)
based on the principle of PCR amplification
amplification is controlled by altering the temperature of solution
the amount of per product amplified correlates with the initial DNA conc
2 categories
end point pCr
real time per (gold std)
end point pcr
measures the amount of amplified product synthesized during per at the end of the reaction
usually use intercalating dyes and fluorescence is measured
other methods as well
compared to standard curve made by samples with known conc
dye in solution emits low fluorescence
emission of fluorescence by binding
dye cycles between an unbound (denatured) and a bound (annealing through extension) state as the reaction progresses and signal intensity increases as the quantity of amplicons increase
real-time per or qPCr
real time, quantitative, not end time
pros
increased sensitivity
large dynamic range (30pg-100ng)
assess dna quality
inhibited DNA samples
degraded DNA samples
high throughput can be automated
multiplexing capabilities
quanititate total human degradation and Y-STRs all at same time
measure small locations and compare to human
quantifies amplified dna during exponential phase of pcr
per developed in early 1990s by Mary mulls and received the Nobel prize in chem 1993
real time per developed by higuchi 1993
happens all in one tube, not opened
good for limiting contamination
analyzes the cycle-cycle change in fluorescence signal resulting from amplification of a target sequence during pcr
uses a fluorescent reporter dye, not an interchelating dye)
tan-man polymerase (applied bio systems) method widely used
the typical per reaction
3 steps
as seen when you amplify targets for str analysis
95C denaturation, dna unwinds into 2 single strands
ssDNA template required for PCR
50-65 annealing, primers anneal to single stranded DNA
primers required
actual temp is determined by primer sequence
72 extension
DNA polymerase incorporates to dNTPs
requires enzyme polymerase
theoretical exponential increase every cycle
real life: limiting reagents
polymerase becomes last, competitive binding with ssDNA strands and primers
Taqman Real time PCR thermal cycling
discovery of thermos aquatics (Taq)
increased efficiency and allowed for automation
two steps for qPCR thermal cycling
denaturation (95)
annealing/extension (60C)
72C elongation is skilled to keep probe annealed
Hot start is added for STR typing
PCR to real time qPCR
need a fluorescent detection system attached to a thermal cycler
need fluorescent markers added to reaction mix
PCR thermocycler
ABI prism 7300 Sequence Detection system
Tungsten Halogen lamp
CCD charged-coupled device, camer
Taq-Man qPCR method
reaction requires DNA template, Taq polymerase enzyme, dNTPs, MgCL2, primers
all above is same as STR PCR and all PCRs
qPCR also requires taqman probes/dye
employs the 5’ exonuclease activity of Taq
Taq-Man qPCR probe
probe is designed to anneal to a target sequence located between the upstream and downstream primers
remember DNA is complimentary and anti-parallel
the probe Tm should be higher than the amplification primer Tm
ensure probe binds to target sequence prior to primer binding and annealing phase
minor groove binder. (MGB) is often linked to 3’ end of probe to allow for shorter probe by increasing the Tm value of the probe
probe is labeled with both a reported fluorescent dye and quencher dyreporter on 5’, quencher on 3’
while probe is intact, the proximity of the two des reduce the fluorescence emitted by the reporter dye
this is called FRET, fluorescent resonance energy transfer)
once probe is cleaved, the reporter dye is released from quencher dye and fluoresces
the intensity of the fluorescence is directly proportional to the amount of target DNA synthesized during the PCR
Taw-man has autocorrect feature which allows it to digest reporter dye from probe
munch up and gets rid of nucleotides in its way
R is on 5’ end
when loading plate for qPCR
column 1&2 will be for standards
50, 5, 0.5, 0.05, 0.005 ng/uL
wide quantification range
qPCR neg control, components of quantification mix
extracted DNA samples and RB (extraction reagents, to look for contamination in extraction step)
2uL of sample will be added to each well
when setting up qPCR
primer mix: contains the target specific primers, dye-labeled probes, and IPC template (internal + control lab made dna that has complimentary sequences for primers and probe, shows everything is working properly)
reaction mix: contains dNTPs buffer, enzyme, passive reference standard, stabilizers
reference standard is the reference for base fluorescent
serial dilution of standards
add up all the samples including std and blanks + 2-3 reactions for pipetting errors
add 8uL of primer mix per sample
10 uL of reaction mix per sample
master mix, 18uL to all sample wells being used
PCR exponential amplification
the final number of copies equals 2^n n=number of cycles if the reaction is working 100% efficiently
x axis is cycle number
y axis is log target DNA fluorescent
linear (theoretical)
not log is exponential
actual curve is different
exponential phase is linear on log
linear phase is slight curve on log
plateau
stage 1
exponential 2n increase, most efficient part of reaction
stage 2
linear increase
no longer 2n increase (limiting reagent, primer, probe, dents, not as efficient)
stage 3
plateau
reaction slows and stops reduced Taq activity, limiting primers and dNTPs, product and primer reannealing competition
product does not have probe, limiting
both product and primer are complimentary
cycle threshold (CT)
arbitrary # chosen to determine total DNA in sample
instrument plots the rate of accumulation of amplified DNA over the course of an entire PCR
greater the initial concentration of target sequences, the fewer cycles required to achieve a particular yield of amplified pdf
CT defined as the number of PCR cycles required to reach an arbitrary threshold of amplification product
initial conc of target sequences determines CT
lower int conc = higher CT
higher int conc = lower CT
measure when sample crosses CT and compare with std curve
known std are run in parallel to samples creating a standard curve
unknown samples are plotted against this curve to determine DNA conc of sample
x axis is log DNA conc
CT is y axis
that is std. curve
efficiency is based on slope of line
agencies have a r^2 limit, >.9
r2 is a measurement of how well the actual data fit to the std curve
quant curve is a negative slope around -3.225
bc greater quantity = lower CT
