Polymerase Chain Reaction

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Polymerase Chain Reaction

• What is PCR
• History of PCR
• How PCR works
• Optimizing PCR
• Fidelity, errors cloning
• PCR primer design
• Application of PCR

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Polymorphism among 32 wheat samples revealed by
AFLP
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Characteristics of AFLP

• dominant marker.
• DNA variation is detected by presence/absence of
  DNA bands due to
• a) presence/absence of restriction sites
• b) additional bases (insertion) between two
  restriction sites are too large

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Advantages

• higher reproducibility compared to RAPD.
• highly polymorphic.

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Think
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PCR Requirements

• Magnesium chloride .5-2.5mM
• Buffer pH 8.3-8.8
• dNTPs 20-200µM
• Primers 0.1-0.5µM
• DNA Polymerase 1-2.5 units
• Target DNA ? 1 µg

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Can I PCR Amplify RNA?

• Not directly the DNA polymerase requires a DNA
  template and will not copy RNA.
• mRNA can first be copied into cDNA using reverse
  transcriptase.
• cDNA is a template for PCR it need not be
  double-stranded.

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Cloning PCR Products

• Products should be ligatable into blunt-ended
  restriction enzyme site.
• Lower than expected efficiency.
• Products are not truly blunt-ended.
• Taq polymerase adds a single nontemplated base
  (usually A) to the 3 end
• NNNNNNNNNNNNNNA
• ANNNNNNNNNNNNNN

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Cloning PCR-generated Fragments

• Generation of compatible ends.
• T/A cloning vectors

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PCR Mutagenesis

• Add restriction sites for cloning
• Generate deletional mutants
• Generate single point mutations

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THE PROBLEM

• QUANTITATION OF mRNA
• northern blotting
• ribonuclease protection assay
• in situ hybridization
• PCR
• most sensitive
• can discriminate closely related mRNAs
• technically simple
• but difficult to get truly quantitative results
  using conventional PCR

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Protocol RT-PCR

• Isolate RNA (total or polyA selected)
• Use RNA to generate cDNA (reverse transcriptase)
• Use cDNA from RT reaction as template for PCR
  amplification.

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RT-PCR vs Real-time RT-PCR

• RT-PCR is endpoint analysis
• Real-time (fluorescent quantitative) PCR is
  continual analysis

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Application of Real-time PCR

• Expression analysis (RNA)
• Identification/quantification of single base pair
  polymorphisms (DNA)
• Genotyping (DNA)
• Exposure monitoring RNA)
• Clinical Screening
• Cancer (DNA)
• Pathogen detection (DNA/RNA)

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Considerations for Fluorescent Real-time RT-PCR

• Primer sets
• Chosing target sequence
• Checking primer sets
• Chosing fluorescent label
• Quantification
• Relative quantification
• Absolute quantification
• Standard curves

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Basics of real time RT-PCRAbundance of mRNA

• Central hypothesis Changes in gene expression
  reflect changes in cell function
• Nearly all physiological changes in animals are
  accompanied by changes in gene expression
• Immune response
• Toxic response
• Pharmacological response

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Outline

• Basics of PCR and real time PCR
• Experimental Design
• Data Analysis
• Application to Drug Development and Design

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Polymerase Chain Reaction

• In vitro method for the amplification of short
  (up to 5000bp) pieces of DNA
• Relies on a thermostable form of DNA polymerase
• Thermus aquaticus

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Polymerase Chain Reaction

• Required reagents
• Template DNA
• Primers
• DNA polymerase
• dNTPs
• Thermocycler

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Polymerase Chain Reaction

• Proper controls
• Water blank
• Concentration curve
• Internal standard control
• Genomic DNA contaminant control

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Internal standard
Gene of interest
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Polymerase Chain Reaction

• Advantages
• Sensitive
• Versatile easy to test new genes
• Primers are inexpensive
• Reliable
• Much more than microarrays for individual
  transcripts
• Standardized competitor templates or standard
  curves
• Allow comparison between expts
• Internal standards
• Addresses variation in tissue starting amounts or
  loading errors

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Polymerase Chain Reaction

• Disadvantages
• Optimizations required
• Annealing temperature
• Number of cycles
• Small order of magnitiude sensitivity for
  detection

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AFLPAmplified Fragment Length Polymorphism
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• Principles of Real-Time Quantitative PCR
  Techniques
• SYBR Green I technique SYBR Green I fluorescence
  is enormously increased upon binding to
  double-stranded DNA. During the extension phase,
  more and more SYBR Green I will bind to the PCR
  product, resulting in an increased fluorescence.
  Consequently, during each subsequent PCR cycle
  more fluorescence signal will be detected.
• Hydrolysis probe technique The hydrolysis probe
  is conjugated with a quencher fluorochrome, which
  absorbs the fluorescence of the reporter
  fluorochrome as long as the probe is intact.
  However, upon amplification of the target
  sequence, the hydrolysis probe is displaced and
  subsequently hydrolyzed by the Taq polymerase.
  This results in the separation of the reporter
  and quencher fluorochrome and consequently the
  fluorescence of the reporter fluorochrome becomes
  detectable. During each consecutive PCR cycle
  this fluorescence will further increase because
  of the progressive and exponential accumulation
  of free reporter fluorochromes.
• Hybridization probes technique In this
  technique one probe is labelled with a donor
  fluorochrome at the 3 end and a second
  adjacent- probe is labelled with an acceptor
  fluorochrome. When the two fluorochromes are in
  close vicinity (15 nucleotides apart), the
  emitted light of the donor fluorochrome will
  excite the acceptor fluorochrome (FRET). This
  results in the emission of fluorescence, which
  subsequently can be detected during the annealing
  phase and first part of the extension phase of
  the PCR reaction. After each subsequent PCR cycle
  more hybridization probes can anneal, resulting
  in higher fluorescence signals.

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What do mRNA levels tell us?

• DNA?mRNA?protein
• Reflect level of gene expression
• Information about cell response
• Protein production (not always)

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quantitative mRNA/DNA analysis
Direct -Northern blotting -In situ
hybridization PCR amplification -Regular
RT-PCR -Real time PCR (Microarrays)
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Why isnt this good enough?
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Whats Wrong With Agarose Gels?

• Low sensitivity
• Low resolution
• Non-automated
• Size-based discrimination only
• Results are not expressed as numbers
• ? based on personal evaluation
• Ethidium bromide staining is not very
  quantitative
• End point analysis

ABI Real-Time PCR vs Traditional PCR (www)
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Endpoint analysis
Different concentrations give similar endpoint
results!
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• Real-time Principles
• based on the detection and quantitation of
• a fluorescent reporter
• In stead of measuring the endpoint we focus on
  the first significant increase in the amount of
  PCR product.
• The time of the increase correlates inversely to
  the initial amount of DNA template

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Polymerization
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For Real Time PCR we need a a specific probe with
a fluorescent reporter.
Probe
Q
R
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When in close contact with the reporter, the
quencer absobes its emission.
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Strand Displacement
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Cleavage
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Polymerization Completed
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Van der Velden. Leukemia 2003 (www)
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• SYBR Green
• (double-stranded DNA binding dye)
• emits a strong fluorescent signal upon binding
  to double-stranded DNA
• nonspecific binding is a disadvantage
• requires extensive optimisation
• longer amplicons create a stronger signal
• Its cheap

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SYBR Green I Chemistry
Polymerization
Forward
Primer
5'
3'
5'
3'
5'
5'
Reverse
Primer
Polymerization completed
5'
3'
5'
3'
5'
5'
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Real-time PCR advantages not influenced by
non-specific amplification amplification can
be monitored real-time no post-PCR processing
of products (high throughput, low contamination
risk) requirement of 1000-fold less RNA than
conventional assays (3 picogram one genome
equivalent) most specific, sensitive and
reproducible
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Housekeeping gene

• Knowing the amount of mRNA in one sample from one
  specific gene does not tell us alot
• You dont know the total amount of mRNA in your
  sample
• You also dont know how much the mRNA level has
  changed compared to other mRNA levels
• Example
• mRNA levels increase 2x after induction
• It is possable that all genexpression in the cell
  has increased
• ?We have to compare the expression of our gene to
  another gene which expression is normally
  constant, a housekeeping gene

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Real-time PCR advantages not influenced by
non-specific amplification amplification can
be monitored real-time no post-PCR processing
of products (high throughput, low contamination
risk) ultra-rapid cycling (30 minutes to 2
hours) wider dynamic range of up to 1010-fold
requirement of 1000-fold less RNA than
conventional assays (3 picogram one genome
equivalent) detection is capable down to a
2-fold change confirmation of specific
amplification by melting curve analysis most
specific, sensitive and reproducible not much
more expensive than conventional PCR (except
equipment cost)
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Real-time PCR disadvantages setting up
requires high technical skill and support high
equipment cost intra- and inter-assay
variation RNA lability DNA contamination (in
mRNA analysis)
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• Principles of Real-Time Quantitative PCR
  Techniques
• SYBR Green I technique SYBR Green I fluorescence
  is enormously increased upon binding to
  double-stranded DNA. During the extension phase,
  more and more SYBR Green I will bind to the PCR
  product, resulting in an increased fluorescence.
  Consequently, during each subsequent PCR cycle
  more fluorescence signal will be detected.
• Hydrolysis probe technique The hydrolysis probe
  is conjugated with a quencher fluorochrome, which
  absorbs the fluorescence of the reporter
  fluorochrome as long as the probe is intact.
  However, upon amplification of the target
  sequence, the hydrolysis probe is displaced and
  subsequently hydrolyzed by the Taq polymerase.
  This results in the separation of the reporter
  and quencher fluorochrome and consequently the
  fluorescence of the reporter fluorochrome becomes
  detectable. During each consecutive PCR cycle
  this fluorescence will further increase because
  of the progressive and exponential accumulation
  of free reporter fluorochromes.
• Hybridization probes technique In this
  technique one probe is labelled with a donor
  fluorochrome at the 3 end and a second
  adjacent- probe is labelled with an acceptor
  fluorochrome. When the two fluorochromes are in
  close vicinity (15 nucleotides apart), the
  emitted light of the donor fluorochrome will
  excite the acceptor fluorochrome (FRET). This
  results in the emission of fluorescence, which
  subsequently can be detected during the annealing
  phase and first part of the extension phase of
  the PCR reaction. After each subsequent PCR cycle
  more hybridization probes can anneal, resulting
  in higher fluorescence signals.

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Schematic diagram comparing three different
fluorescence-monitoring systems for DNA
amplification. System A uses dsDNA-specific dyes
(F) such as SYBR"Green I, which increase in
fluorescence when bound to accumulating
amplification product. System B uses
dual-labelled probes and depends on the
5'-exonuclease activity of the polymerase to
separate donor (D) and acceptor (A) by
hydrolysis. Donor fluorescence is increased by
removing acceptor quenching. System C depends on
the independent hybridization of adjacent donor
(D) and acceptor (A) probes. Their approximation
increases resonance energy transfer from the
donor to the acceptor. Other symbols are "hv" for
excitation light and "x" for a 3'-phosphate.
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TaqMan Probes FRET Förster/fluorescence
resonance energy transfer DNA Polymerase 5'
exonuclease activity Tm value 100 C higher
than primers runs of identical nucleotides (no
consecutive Gs) GC content 30-80 more Cs
than Gs no G at the 5' end
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FRET Förster/fluorescence resonance energy
transfer
ABI Real-Time PCR vs Traditional PCR (www)
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DNA Polymerase 5' Exonuclease Activity
Mocellin et al. Trends Mol Med 2003 (www)
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The TaqMan 5 Exonuclease Assay In addition to
two conventional PCR primers, P1 and P2, which
are specific for the target sequence, a third
primer, P3, is designed to bind specifically to a
site on the target sequence downstream of the P1
binding site. P3 is labelled with two
fluorophores, a reporter dye (R) is attached at
the 5 end, and a quencher dye (D), which has a
different emission wavelength to the reporter
dye, is attached at its 3 end. Because its 3
end is blocked, primer P3 cannot by itself prime
any new DNA synthesis. During the PCR reaction,
Taq DNA polymerase synthesizes a new DNA strand
primed by P1 and as the enzyme approaches P3, its
5 3 exonuclease activity processively degrades
the P3 primer from its 5 end. The end result is
that the nascent DNA strand extends beyond the P3
binding site and the reporter and quencher dyes
are no longer bound to the same molecule. As the
reporter dye is no longer in close proximity to
the quencher, the resulting increase in reporter
emission intensity is easily detected.
Human Molecular Genetics 2. NCBI Books (www)
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TaqMan Primers equal Tm (58-600 C) 15-30
bases in length GC content 30-80 no runs
of four or more Gs (any nucleotide) no more
than two GC at the 3 end no G at the 5' end
amplicon size 50-150 bp (max 400) span
exon-exon junctions in cDNA
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SYBR Green (double-stranded DNA binding dye)
emits a strong fluorescent signal upon binding to
double-stranded DNA nonspecific binding is a
disadvantage requires extensive optimization
requires melting point curve determination
longer amplicons create a stronger signal may
be multiplexed when coupled with melting curve
analysis
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Fluoresces when bound to dsDNA
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SYBR Green (1) At the beginning of
amplification, the reaction mixture contains the
denatured DNA, the primers, and the dye. The
unbound dye molecules weakly fluoresce, producing
a minimal background fluorescence signal which is
subtracted during computer analysis. (2) After
annealing of the primers, a few dye molecules can
bind to the double strand. DNA binding results in
a dramatic increase of the SYBR Green I molecules
to emit light upon excitation. (3) During
elongation, more and more dye molecules bind to
the newly synthesized DNA. If the reaction is
monitored continuously, an increase in
fluorescence is viewed in real-time. Upon
denaturation of the DNA for the next heating
cycle, the dye molecules are released and the
fluorescence signal falls.
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When to Choose SYBR Green
Assays that do not require specificity of probe
based assays. Detection of 1000s of molecules
General screening of transcripts prior to moving
to probe based assays When the PCR system is
fully optimized -no primer dimers or non-specific
amplicons, e.g. from genomic DNA
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When Not to Choose SYBR Green
Allelic discrimination assays (not an absolute
one) Multiplex reactions (not an absolute
one) Amplification of rare transcripts Low
level pathogen detection
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Real-Time Principles Three general methods for
the quantitative detection 1. Hydrolysis
probes (TaqMan, Beacons, Scorpions) 2.
Hybridization probes (Light Cycler) 3.
DNA-binding agents (SYBR Green)
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Molecular Beacons
Mocellin et al. Trends Mol Med 2003 (www)
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Real-Time Principles Three general methods for
the quantitative detection 1. Hydrolysis
probes (TaqMan, Beacons, Scorpions) 2.
Hybridization probes (Light Cycler) 3.
DNA-binding agents (SYBR Green)
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Scorpions
Bustin SA. J Mol Endocrinol 2002 (www)
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Scorpions
Bustin SA. J Mol Endocrinol 2002 (www)
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Threshold Cycle threshold cycle or the CT
value is the cycle at which a significant
increase in DRn is first detected it is the
parameter used for quantitation CT value of 40
or more means no amplification and cannot be
included in the calculations
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What is CT?
The Amplification Plot contains valuable
information for the quantitative measurement of
DNA or RNA. The Threshold line is the level of
detection or the point at which a reaction
reaches a fluorescent intensity above background.
The threshold line is set in the exponential
phase of the amplification for the most accurate
reading. The cycle at which the sample reaches
this level is called the Cycle Threshold, CT.
These two values are very important for data
analysis using the 5 nuclease assay.
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Albumin (ALB) gene dosage by real-time
PCR Laurendeau et al. Clin Chem 1999 (www)
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DRn Rn is the Rn value of a reaction
containing all components (the sample of
interest) Rn- is the Rn value detected in NTC
(baseline value) DRn is the difference between
Rn and Rn-. It is an indicator of the magnitude
of the signal generated by the PCR DRn is
plotted against cycle numbers to produce the
amplification curves and gives the CT value
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What is DRn?
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What is DRn?
(www)
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Endogenous/Internal Control (Normalization)
usually an abundantly and constantly expressed
housekeeping gene most commonly used ones are
the least reliable ones best to run a validity
test for the selected endogenous control
combination may/should be used
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Endogenous Control Selection
Sabek et al. Transplantation 2002 (www)
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Efficiency The slope of the log-linear phase is
a reflection of the amplification efficiency The
efficiency of the reaction can be calculated by
the following equation Eff10(-1/slope) 1. The
efficiency of the PCR should be 90-100 (ideal
slope 3.3) A number of variables can affect
the efficiency of the PCR. These factors can
include length of the amplicon, secondary
structure, and primer design, to name a few
Approximation vs Pfaffl method (Efficiency
Determination)
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Using the PCR Equation
Xn
Xn X0(1 E)n
Xn PCR product after cycle n X0 initial copy
number E amplification efficiency n cycle
number
X0
cycle number
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Effect of Amplification Efficiency
Xn X0(1E)n
Case 1 E 0.9
Case 2 E 0.8
Xn 100 (10.9)30
Xn 100 (10.8)30
Xn 2.3 x 1010
Xn 4.6 x 109
Result A difference of 0.1 in amplification effici
encies created a five-fold difference in
the final ratio of PCR products after 30 cycles
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Determination of real-time PCR efficiencies of
reference gene (Gst), target gene 1 (TyrA) and
target gene 2 (PyrB). CP cycles versus cDNA
(reverse transcribed total RNA) concentration
input were plotted to calculate the slope (mean
SD n 3). The corresponding real-time PCR
efficiencies were calculated according to the
equation E 101/slope
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If the CT values for each of the dilutions are
plotted against concentrations, the result should
be a linear graph with a high correlation
coefficient (gt 0.99). The slope of this graph is
also a measure of efficiency, and can be readily
used to calculate efficiency - this is done by
most software (iCycler, for example).
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Nigel Walker, NIEHS (www)
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Nigel Walker, NIEHS (www)
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Assay Validation Test primer pairs in all
combinations with the probe with a known template
(plasmid clone, sDNA, RNA) Use standard assay
conditions 300-400 nM primers 100 nM probe, 3
mM MgCl2 Choose the primer pair that gives the
highest DRn and the lowest CT Make a dilution
of a template, either sDNA, sRNA or total RNA for
a standard curve Correlation coefficient of
the standard curve gt 0.99? If the slope of the
standard curve of the best primer pair is around
-3.5 increase the MgCl2 to 5 mM If the slope
is higher than -3.6, change primers An ideal
assay will have a slope of -3.3
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One-Step or Two-Step PCR one-step real-time
RT-PCR performs reverse transcription and PCR in
a single buffer system and in one tube in
two-step RT-PCR, these two steps are performed
separately in different tubes
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The really big deal

• Isolation of DNA polymerase from Thermus
• aquaticus (Mullis and Faloona 1987)
• T. aquaticus is a hot springs bacterium
• Therefore, has thermally stable DNA
• polymerase (e.g. not killed by heat)

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Importance of T. aquaticus

• Extension at 72 C Most polymerases are
• active at 45 C or below (e.g. normal
• body/environmental temps)-temps below
• normal annealing temps-results in nonspecific
• binding of primers
• 2. From Bacterium that lives in near boiling
• water Most polymerase inactivated by high
• temperatures-initially had to add new
• polymerase each cycle

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Colony PCR

• CPSC265 Class 8

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Cloning

• Cloning is the way in which we can take a single
  molecule, and make lots of bacterial cells that
  contain an identical molecule.
• These cells are clones, hence the name
• This used to be the only way to amplify DNA. It
  is still by far the most accurate.

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Plasmid vectors circular, autonomous bacterial
DNA
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The vector is made with a T overhang
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Taq polymerase leaves an A overhang

• Taq is the thermostable DNA polymerase from
  Thermus aquaticus we used for PCR.
• When Taq synthesizes a new strand, it always puts
  an extra A at the end
• This can be useful, but note other polymerases
  do not do this, they leave blunt ends. Only Taq
  polymerase leaves A overhangs. Blunt end
  vectors do not work with Taq, we need a T
  overhang.

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DNA ligase

• Repairs gaps in the sugar-phosphate backbone of
  DNA
• Creates phosphodiester bonds
• Does not do anything with the bases

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Transformation of bacteria

• Two main methods for transformation
• Chemical / Heat Shock
• As done in last practical, this method gets DNA
  into the cell by making them porous using CaCl2
  and a 42 C heat treatment
• Electroporation
• Makes cells porous using high-voltage electricity

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Imperfect science

• Most of the plasmid / insert combinations will
  not ligate
• Most of the bacteria will not be transformed
• We only need one molecule to get into one
  bacterium to make one colony.

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PCR from clones

• Often clones will religate containing any old DNA
  (eg primer dimers)..
• The DNA can go in in either orientation
• We can use the PCR to tell which colonies have
  the insert we want, and which orientation it is
  in.

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Some population genetics software

• Microsatellite toolkit Excel plug-in for
  creating Arlequin, FSTAT and Genepop files.
• Microchecker Estimate null allele
  frequency. Adjust allele frequencies.
• Arlequin HW equilibrium,
  Linkage Disequilibrium, Fst, exact test of
• differentiation,
  Amova, Mantel test
• FSTAT Allelic richness, Fst
  per locus (to check contribution of each
• locus to
  observed pattern of differentiation)
• Structure, BAPS Population structuring,
  population assignment.
• Migrate Estimates of effective population size
  and migration rates
• Bottleneck Check for very recent
  population bottlenecks