tools for molecular biology amplification. the pcr reaction is a way to quickly drive the...
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tools for Molecular Biology
Amplification
The PCR reaction is a way to quickly drive the exponential amplification of a small piece of DNA.
PCR is a 3 step process• Denaturation of the target DNA
• Annealing of your gene specific primers
• Elongation of the target DNA by a Heat Stabile DNA Polymerase
• Amplification progresses exponentially so that the final number of copies equals 2n (n=number of cycles)
The PCR Reaction
The PCR Reaction
5’
5’
3’
3’
d.NTPs
Thermal Stable DNA Polymerase
Primers5’
3’5’
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3’
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3’
5’
3’5’
3’
Add Master Mix and Sample
Denaturation
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3’
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5’
3’5’
3’
Annealing
Add to Reaction Tube
Extension
5’ 3’
5’3’5’ 3’
5’3’
Extension Continued
5’ 3’
5’3’5’
5’
Taq
Taq
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5’3’
Taq
Taq5’
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Repeat
The PCR Reaction
THE PCR REACTION
5’3’5’ 3’
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Cycle 2
4 Copies
Cycle 3
8 Copies
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3’
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PCR - Powerful Tool!!
PCR technology is an essential tool for Molecular Biology
PCR allows rapid and reproducible amplification of a specific sequence of DNA
PCR technology is responsible for accelerating Genetic Discoveries
Real Time PCR incorporates the ability to directly measure and
quantify the reaction while amplification is taking place.
What is Real Time PCR?
Cycle #
Log
Tar
ge
t DN
A
Theoretical
Amplification is exponential, but the exponential increase is limited:
Real-Time PCR allows us to ‘see’ the exponential phase so we can calculate how much we started with.
A linear increase follows exponential Eventually plateaus
Real Life
ThresholdThreshold
CCTT
Reality vs. Theory
Threshold Cycle, Ct, of the same 96 replicates shows nearly identical
values
0
0.4
0.8
1.2
1.6
2
0 10 20 21 30 40
Threshold
Baseline
Sample
CCTT
What is Threshold Cycle (CT)?
Correlates strongly with the starting copy number If you have twice the template, you get to Ct one
cycle earlier If you have half the template, you reach Ct one
cycle later
The threshold cycle, Ct
Threshold Cycle, CT
Detection of 125 genomic equivalents from 250. Two-fold serial dilutions of human genomic DNA (gDNA) from 125 to 16,000 genomic equivalents were assayed for -actin.
r = is a measure of how well the actual r = is a measure of how well the actual data fit to the standard curve.data fit to the standard curve.
= (explained variation/total variation)= (explained variation/total variation)
The slope of the standard curve can be The slope of the standard curve can be directly correlated to the efficiency of the directly correlated to the efficiency of the
reactions:reactions:
Efficiency (Efficiency () = [10) = [10(-1/slope)(-1/slope) ] - 1 ] - 1
Threshold Cycle, CT, can be used to generate standard curves
Copy Number vs. Ct - Standard Curve
y = -3.3192x + 39.772
R2 = 0.9967
0
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0 1 2 3 4 5 6 7 8 9 10 11
Log of copy number (10n)
Ct
Threshold Cycle, Ct, is a reliable indicator of initial
copy number
What Detection Strategies can we use?
Intercalating Dyes Intercalating Dyes are inexpensive compared to
hybridization probes. - general confirmation of amplification - NON
SPECIFIC Russ Higuchi demonstrated the key principle of
Real Time PCR using Ethidium Bromide - EtBr fluoresces 25 times more brightly when EtBr fluoresces 25 times more brightly when
bound to dsDNA bound to dsDNA SYBR Green, a more sensitive intercalating dye
is an even more attractive approach SYBR Green fluoresces 200 times more SYBR Green fluoresces 200 times more
brightly when bound to dsDNAbrightly when bound to dsDNA
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’
Add Master Mix and Sample
Denaturation
5’
3’
5’
3’
5’
3’
5’
3’
5’
3’5’
3’
Annealing
Reaction Tube
Intercalating Dyes
Intercalation Dyes
Taq ID
Extension
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5’3’5’ 3’
5’3’
Extension ContinuedApply Excitation
Wavelength
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Taq
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Taq5’
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Repeat
Intercalating Dyes
ID ID
ID IDID
ID ID ID
ID ID
Hybridization Probes
• Cleavage Based Assay– TaqMan Assays
• Displaceable Probe Assays– Molecular Beacons
– Dual oligo FRET probes
• Probes incorporated directly into the primers– Amplifluor
– Scorpions
Today Hybridization Probe Strategies fall into three main categories:
TaqManTM
5’
5’
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d.NTPs
Thermal Stable DNA Polymerase
Primers5’
3’5’
3’
5’
3’
5’
3’
5’
3’
5’
3’
5’
3’5’
3’
Add Master Mix and Sample
Denaturation
5’
3’
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3’
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5’
3’5’
3’
Annealing
Reaction Tube
Taq
5’ 3’
R Q
Probe
5’ 3’
R Q
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TaqManTM
5’3’
RQ
Extension Step
5’ 3’
1. Strand DisplacementTaq3’
Q
R
5’
5’ 3’3’
Q Taq
R
5’ 2. Cleavage
3. PolymerizationComplete5’ 3’
QTaqR
3’ 5’
4. Detection5’ 3’
3’
QTaqR
5’
R
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’
Add Master Mix and Sample
Annealing
Reaction Tube
Molecular Beacons
Denaturation
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3’
5’
3’5’
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Taq
5’ 3’
R Q
R Q
MolecularBeacon
5’ 3’5’3’
Extension Step5’ 3’
1. Strand DisplacementTaq5’
2. PolymerizationComplete
Probe Silent
Molecular Beacons
5’3’
RQ
Detection
5’3’
RQ
5’ 3’
3’ 5’
TaqR Q
MolecularBeacon
FRET Probes
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3’
D R
5’ Detection
Extension Step5’ 3’
1. Strand DisplacementSystem Silent
2. PolymerizationComplete
System Silent
5’ 3’
3’ 5’
Taq
3’
D
R1-5 bases
D
R
Taq5’
R
5’
Primer Based
3’5’
R Q
Heat
Incorporation
R Q
Primer Based
5’
Extension 23’5’
R
Q
5’ 3’ 5’R Q Annealing/Extension 13’
QR
3’
5’ 3’
5’
Detection
Melt Curve Analysis• This type of analysis measures the decrease in fluorescence as the temperature
slowly increases. • The decrease in fluorescence is caused by the probe dissociating from the target. • Since changes in sequence result in changes in Tm, one can detect mutations by
comparing the amount of fluorescence observed at different Tm.• Mutation detection (SNPs) is not the only application for Melt Curve Analysis• Probe or primer and target melt characterization, and validation of reactions with
SYBR Green are other popular uses for Melt Curve Analysis.
Melt Curve: what is it?Melt Curve: what is it?
Discriminates by Melting Temperature (Tm) - the temperature at which 50% of the DNA molecules separate into two strands - or “melts” apart
Tm is dependent on: sequence (G/C content) length complementarity
Fluorescence vs. Temperature
change in fluorescence
single amplified product
Tm
Yes We Still Run Gels!!!
Validation with SYBR Green
Melt CurveCheck specificity of the reactionCheck specificity of the reaction
Melt curve showing two amplified products
Melt Curve
• Tests for specificity in all samples
• Discriminates by melting temperature
• Not as high resolution as running a gel
• Run melt curve followed by gel of representative samples