tools%20for%20Molecular%20Biology%20Amplification

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tools for Molecular BiologyAmplification
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The PCR Reaction

• 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 (nnumber of
  cycles)

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The PCR Reaction
d.NTPs
Primers
Add Master Mix and Sample
Thermal Stable DNA Polymerase
Add to Reaction Tube
Denaturation
Annealing
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The PCR Reaction
Extension
Extension Continued
Repeat
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THE PCR REACTION
Cycle 2 4 Copies
Cycle 3 8 Copies
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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

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What is Real Time PCR?
Real Time PCR incorporates the ability to
directly measure and quantify the reaction while
amplification is taking place.
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Reality vs. Theory
Amplification is exponential, but the exponential
increase is limited

• A linear increase follows exponential
• Eventually plateaus

Theoretical
Log Target DNA
Real-Time PCR allows us to see the exponential
phase so we can calculate how much we started
with.
CT
Cycle
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Threshold Cycle, Ct, of the same 96 replicates
shows nearly identical values
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What is Threshold Cycle (CT)?
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The 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

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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 b-actin.
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Threshold Cycle, CT, can be used to generate
standard curves
r is a measure of how well the actual data fit
to the standard curve. (explained
variation/total variation)
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Threshold Cycle, Ct, is a reliable indicator of
initial copy number
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What Detection Strategies can we use?
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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 bound
  to dsDNA
• SYBR Green, a more sensitive intercalating dye is
  an even more attractive approach
• SYBR Green fluoresces 200 times more brightly
  when bound to dsDNA

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Intercalating Dyes
d.NTPs
Primers
Intercalation Dyes
Add Master Mix and Sample
Thermal Stable DNA Polymerase
Reaction Tube
l
Denaturation
Annealing
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Intercalating Dyes
Extension
Extension Continued Apply Excitation Wavelength
Repeat
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Hybridization Probes
Today Hybridization Probe Strategies fall into
three main categories

• Cleavage Based Assay
• TaqManä Assays
• Displaceable Probe Assays
• Molecular Beacons
• Dual oligo FRET probes
• Probes incorporated directly into the primers
• Amplifluor
• Scorpions

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TaqManTM
d.NTPs
Primers
Add Master Mix and Sample
Thermal Stable DNA Polymerase
Reaction Tube
Denaturation
l
Annealing
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TaqManTM
Extension Step
1. Strand Displacement
2. Cleavage
3. Polymerization Complete
l
4. Detection
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Molecular Beacons
Reaction Tube
Annealing
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Molecular Beacons
l
Detection
Extension Step
1. Strand Displacement
2. Polymerization Complete Probe Silent
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FRET Probes
l
Detection
1-5 bases
Extension Step
1. Strand Displacement System Silent
2. Polymerization Complete System Silent
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Primer Based
l
Heat
Incorporation
l
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Primer Based
Extension 2
l
Detection
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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.

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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

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Fluorescence vs. Temperature
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Validation with SYBR Green
Yes We Still Run Gels!!!
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Melt CurveCheck specificity of the reaction
Melt curve showing two amplified products
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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