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REAL TIME QUANTIFICATION. PCR reaction to be visualized “in real time” as the reaction progresses to quantify the amount of DNA in the sample at the start of the reaction. 5’. 3’. 5’. 3’. 3’. 3’. 3’. 3’. 3’. 3’. 5’. 5’. 5’. 5’. 5’. 5’. 5’. 5’. 5’. 5’. 5’. 5’. 3’. 3’. 3’. - PowerPoint PPT Presentation
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•PCR reaction to be visualized “in real time” as the reaction progresses
•to quantify the amount of DNA in the sample at the start of the reaction
REAL TIME QUANTIFICATION
5’5’3’
3’
d.NTPs
Thermal Stable DNA Polymerase
Primers5’
3’5’
3’
5’
3’
5’3’
5’
3’
5’
3’
5’
3’5’
3’
Denaturation
5’
3’
5’
3’
5’
3’
5’3’
5’
3’5’
3’
Annealing
Add to Reaction Tube
THE REACTION
Extension
5’ 3’
5’3’5’ 3’
5’3’
Extension Continued
5’ 3’
5’3’5’
5’
Taq
Taq
3’
5’3’
Taq
Taq5’
5’
Repeat
5’3’5’ 3’
5’3’5’
3’
5’5’3’
3’
5’3’5’ 3’
Cycle 2
4 Copies
Cycle 3
8 Copies
5’3’5’
3’
5’3’5’ 3’
5’3’5’ 3’
5’3’5’ 3’
5’3’5’ 3’
5’3’3’5’
5’3’5’ 3’
5’3’5’ 3’
0
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
0 5 10 15 20 25 30 35 40
2*10=1024
4*5=1024
0
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
0 5 10 15 20 25 30 35 40
Ct values are directly related to the starting quantity of DNA, by way of the formula:
Quantity = 2^Ct
0
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
0 5 10 15 20 25 30 35 40
23 2528
Ct Values:
Copy Number vs. Ct - Standard Curve
y = -3.3192x + 39.772
R2 = 0.9967
0
5
10
15
20
25
30
35
40
0 1 2 3 4 5 6 7 8 9 10 11
Log of copy number (10n)
C t
As shown by Higuchi et al.2, a plot of the log of initial target copy number for a set of standards versus CT is a straight line.
Effect of Limiting Reagents
CT is a more reliable measure of starting copy number than an endpoint measurement
the rate of target amplification decreases until a plateau is reached
1. double stranded DNA binding dye: SYBR GREEN ®
2. fluorogenic probes: Taqman ® Probes
Detection of PCR product accumulation
SYBR Green real time
Extension
5’ 3’
5’3’5’ 3’
5’3’
Apply ExcitationWavelength
5’ 3’
5’3’5’
5’
Taq
Taq
3’
5’3’
Taq
Taq5’
5’
Repeat
ID ID
ID IDID
ID ID IDID ID
l l l
ll
5700 system: able to use an internal reference dye (ROX)
normalize for non-PCR-related, well-to-well fluctuations in fluorescence
INTERNAL REFERENCE DYE
fluorescence readings taken at 95 ºC in the baseline region and is
essential for reproducible results.
Fluorophores: 6-carboxyfluorescein, acronym: FAM,
tetrachlorofluorescein, acronym: TET
Quenchers: tetramethylrhodamine, acronym: TAMRA,
minor groove binder, MGB
fluorophore covalently attached to the 5’-end of the oligonucleotide probe and
a quencher at the 3’-end.
PROBE BASED real time
PROBE BASED real time
the 5' to 3' exonuclease activity of the polymerase degrades the probe that has annealed to the template.
Degradation of the probe releases the fluorophore from it and breaks the close proximity to the quencher, thus relieving the quenching effect and allowing fluorescence of the fluorophore.
Hence, fluorescence detected in the quantitative PCR thermal cycler is directly proportional to the fluorophore released and the amount of DNA
1. Template preparation and quality
2. Determining concentration and purity of nucleic acids
• Purity of nucleic acid templates is particularly important for real-time PCR, since
contaminants can interfere with fluorescence detection. more sensitive to impurities
such as proteins, phenol/chloroform, salts, and EDTA
• removal of genomic DNA
3. Storage of DNA and RNA
•kits, no degradation is detectable for at least 1 year
•Purified RNA should be stored at -20°C or -70°C in RNase-free water, under slightly
basic conditions (e.g., Tris•Cl, pH 8.0) acidic conditions can cause hydrolysis of DNA.
•Diluted solutions of nucleic acids (e.g., dilution series used as standards) should be
stored in aliquots and thawed once only.
•storage of aliquots in siliconized tubes where possible. This avoids adsorption of
nucleic acids to the tube walls, which would reduce the concentration of nucleic acids
in solution. For long-term storage of DNA samples, QIAsafe DNA Tubes and 96-Well
Plates, which provide stable, room-temperature storage.
4. RT-PCR
1. Assay design
A. Automated
B. Self designed
One-step RT-PCR combines the first-strand cDNA synthesis (reverse transcription) reaction
and PCR reaction in the same tube,
easier processing of large numbers of samples,
and helps minimize carryover contamination, since tubes are not opened between cDNA
synthesis and amplification. By amplifying the entire cDNA sample, one-step RT-PCR can
provide greater sensitivity—from as little as 0.01 pg total RNA.
One-step reactions allow for the use of sequence-specific primers only.
Two-step PCR begins with the reverse transcription of either total RNA or poly(A)+
RNA into cDNA using a reverse transcriptase.
When performing two-step RT-PCR, you have the option of using either oligo(dT),
random hexamer, or gene-specific primers,
then PCR is performed with either Platinum® Taq DNA Polymerase, Platinum® Taq
DNA Polymerase High Fidelity, or your choice of PCR enzyme.
2. One step vs two step RT-PCT
2. One step RT-PCR
Component
gDNA Wipeout Buffer
Quantiscript Reverse Transcriptase
Quantiscript RT Buffer
RT Primer Mix
Component
HotStarTaq DNA Polymerase
QuantiTect SYBR Green RT-PCR Buffer
dNTP mix
SYBR Green I dye
ROX dye
Omniscript and Sensiscript Reverse Transcriptases
QuantiTect SYBR® Green RT-PCR KitQuantiTect Reverse Transcription Kit
Two step RT-PCR
3. Handling and storing primers
Storage
buffer
Lyophilized primers should be dissolved in a small volume of low-salt buffer to
give a concentrated stock solution (e.g., 100 uM). We recommend using TE (10
mM Tris•Cl, 1 mM EDTA, pH 8.0) for standard primers.
Storage Primers should be stored in TE in small aliquots at -20°C. Standard primers are
stable under these conditions for at least 1 year. Repeated freeze-thaw cycles
should be avoided, since they may lead to degradation.
Dissolving
primers
Before opening a tube containing lyophilized primer, spin the tube briefly to
collect all material at the bottom of the tube. To dissolve the primer, add the
required volume of TE, mix, and leave for 20 minutes to allow the primer to
completely dissolve. Mix again and determine the concentration by
spectrophotometry as described below.
We do not recommend dissolving primers in water. They are less stable in water
than in TE and some may not dissolve easily in water.
Primer
quality
Select company carefully
1. No template control (NTC)
All quantification experiments should include an NTC, containing all the
components of the reaction except for the template. This enables detection of
contamination.
2. No RT control
All RT-PCR experiments should include a negative control to test for
contaminating DNA.
3. Positive control
CONTROLS
ABSOLUTE AND RELATIVE QUANTIFICATION
A standard curve (plot of CT value/crossing point against log of amount of
standard) is generated using different dilutions of the standard. The target and
each of the standards are amplified in separate tubes. The CT value of the target is
compared with the standard curve, allowing calculation of the initial amount of
the target. It is important to select an appropriate standard for the type of
nucleic acid to be quantified.
Absolute quantification
Absolute quantification
Relative quantification
With this method, the amounts of the
target genes ....
and the reference gene......house keeping gene
The expression level of the reference gene must not vary under different experimental conditions, or in
different states of the same tissue (e.g., "disease" versus "normal" samples).
The quantification procedure differs depending on whether the target genes and the reference gene are
amplified with comparable or different efficiencies. For determination of PCR efficiency,
The typical method to analyze real-time PCR data is the ΔΔCt method. Its mathematics assumes that the real-time PCR assay has a 100% amplification efficiency. As the primers deviate from this ideal, the error in the fold difference increases exponentially. The traditional method for determining amplification efficiency requires a calibration curve. Serially dilute an artificial template of known concentration. Plot the Ct values versus the initial amounts of input material on a semi-log10 plot, fit the data to a straight line (Fig) and calculate the slope. The closer the slope is to -3.33, the closer the amplification efficiency is to the 100% ideal.
MELTING CURVES
To carry out melting curve analysis, the temperature is increased very slowly from a low
temperature (e.g., 65°C) to a high temperature (e.g., 95°C). At low temperatures, all PCR
products are double stranded, so SYBR Green I binds to them and fluorescence is high, whereas
at high temperatures, PCR products are denatured, resulting in rapid decreases in fluorescence.
The fluorescence is measured continuously as the temperature is increased and plotted against
temperature. A curve is produced, because fluorescence decreases slightly through the lower end
of the temperature range, but decreases much more rapidly at higher temperatures as the
melting temperatures of nonspecific and specific PCR products are reached. The detection
systems calculate the first derivatives of the curves, resulting in curves with peaks at the
respective Tms. Curves with peaks at a Tm lower than that of the specific PCR product indicate the
formation of primer-dimers, while diverse peaks with different Tms or plateaus indicate production
of nonspecific products or a smear
Real-Time PCR has become a cornerstone of molecular biology:
• Gene expression analysis– Cancer research– Drug research
• Disease diagnosis– Viral quantification
• Food testing– Percent GMO food
• Transgenic research– Gene copy number
TaqMan probe-based assays are widely used in quantitative PCR in research and medical laboratories:Gene expression assaysPharmacogenomicsHuman Leukocyte Antigen (HLA) genotypingDetermine the viral load in clinical specimens (HIV, Hepatitis)Bacterial Identification[7] assaysDNA quantificationSNP genotypingVerification of microarray results
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