PCR

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PCR

• Mr. S.Ghosh
• MITS
• Division of Biotechnology

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Laboratory Tools and Techniques
The methods used by molecular biologists to study
DNA have been developed through adaptation of the
chemical reactions and biological processes that
occur naturally in cells Many of the enzymes
that copy DNA, make RNA from DNA, and synthesize
proteins from an RNA template were first
characterized in bacteria. This basic research
has become fundamental to our understanding of
the function of cells and have led to immense
practical applications for studying a gene and
its corresponding protein. As science advances,
so do the number of tools available that are
applicable to the study of molecular genetics.
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PCR
The Polymerase Chain Reaction (PCR) provides an
extremely sensitive means of amplifying
relatively large quantities of DNA First
described in 1985, Nobel Prize for Kary Mullis in
1993 The technique was made possible by the
discovery of Taq polymerase, the DNA polymerase
that is used by the bacterium Thermus aquaticus
that was discovered in hot springs The primary
materials, or reagents, used in PCR are - DNA
nucleotides, the building blocks for the new DNA
- Template DNA, the DNA sequence that you want
to amplify - Primers, single-stranded DNAs
between 20 and 50 nucleotides long
(oligonucleotides) that are complementary to a
short region on either side of the template DNA
- DNA polymerase, a heat stable enzyme that
drives, or catalyzes, the synthesis of new DNA
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PCR
The cycling reactions There are three major
steps in a PCR, which are repeated for 20 to 40
cycles. This is done on an automated Thermo
Cycler, which can heat and cool the reaction
tubes in a very short time. Denaturation at
around 94C During the denaturation, the
double strand melts open to single stranded DNA,
all enzymatic reactions stop (for example the
extension from a previous cycle). Annealing at
around 54C Hydrogen bonds are constantly
formed and broken between the single stranded
primer and the single stranded template. If the
primers exactly fit the template, the hydrogen
bonds are so strong that the primer stays
attached Extension at around 72C The bases
(complementary to the template) are coupled to
the primer on the 3' side (the polymerase adds
dNTP's from 5' to 3', reading the template from
3' to 5' side, bases are added complementary to
the template)
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PCR
The different steps of PCR
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PCR
Exponential increase of the number of copies
during PCR
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PCR
Every cycle results in a doubling of the number
of strands DNA present After the first few
cycles, most of the product DNA strands made are
the same length as the distance between the
primers The result is a dramatic amplification
of a the DNA that exists between the primers. The
amount of amplification is 2 raised to the n
power n represents the number of cycles that are
performed. After 20 cycles, this would give
approximately 1 million fold amplification. After
40 cycles the amplification would be 1 x 1012
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PCR and Contamination
The most important consideration in PCR is
contamination Even the smallest contamination
with DNA could affect amplification For example,
if a technician in a crime lab set up a test
reaction (with blood from the crime scene) after
setting up a positive control reaction (with
blood from the suspect) cross contamination
between the samples could result in an erroneous
incrimination, even if the technician changed
pipette tips between samples. A few blood cells
could volitilize in the pipette, stick to the
plastic of the pipette, and then get ejected into
the test sample Modern labs take account of this
fact and devote tremendous effort to avoiding
cross-contamination
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Optimizing PCR protocols
PCR can be very tricky
While PCR is a very powerful technique, often
enough it is not possible to achieve optimum
results without optimizing the protocol Critical
PCR parameters - Concentration of DNA template,
nucleotides, divalent cations (especially
Mg2) and polymerase - Error rate of the
polymerase (Taq, Vent exo, Pfu) - Primer design
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Primer design
General notes on primer design in PCR
Primer selection Critical variables are -
primer length - melting temperature (Tm) -
specificity - complementary primer sequences -
G/C content - 3-end sequence
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Primer design
Specificity Primer specificity is at least
partly dependent on primer length there are many
more unique 24 base oligos than there are 15
base pair oligos Probability that a sequence of
length n will occur randomly in a sequence of
length m is Example the mtDNA genome has about
20,000 bases, the probability of
randomly finding sequences of length n is n
Pn 5 19.52 10 1.91 x
10-2 15 1.86 x 10-5
P (m n 1) x (¼)n
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Primer design

• Complementary primer sequences
• primers need to be designed with absolutely no
  intra-primer homology beyond 3 base pairs. If a
  primer has such a region of self-homology, snap
  back can occur
• - another related danger is inter-primer
  homology partial homology in the middle
  regions of two primers can interfere with
  hybridization. If the homology should occur at
  the 3' end of either primer, primer dimer
  formation will occur

• G/C content
• ideally a primer should have a near random mix
  of nucleotides, a 50 GC content
• there should be no PolyG or PolyC stretches that
  can promote non-specific annealing

3-end sequence - the 3' terminal position in
PCR primers is essential for the control of
mis-priming - inclusion of a G or C residue at
the 3' end of primers helps to ensure correct
binding (stronger hydrogen bonding of G/C
residues)
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Primer design
Melting temperature (Tm)
- the goal should be to design a primer with an
annealing temperature of at least 50C
- the relationship between annealing temperature
and melting temperature is one of the Black
Boxes of PCR
- a general rule-of-thumb is to use an annealing
temperature that is 5C lower than the melting
temperature
- the melting temperatures of oligos are most
accurately calculated using nearest neighbor
thermodynamic calculations with the formula
Tm H S R ln (c/4) 273.15 C
16.6 log 10 K (H is the enthalpy, S is the
entropy for helix formation, R is the molar gas
constant and c is the concentration of primer)
- a good working approximation of this value can
be calculated using the Wallace formula
Tm 4x (CG)
2x (AT) C
- both of the primers should be designed such
that they have similar melting temperatures. If
primers are mismatched in terms of Tm,
amplification will be less efficient or may not
work the primer with the higher Tm will
mis-prime at lower temperatures the primer with
the lower Tm may not work at higher
temperatures.
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The PCR ProcessReaction Components

• Typical components of a PCR include
• DNA the template used to synthesize new DNA
  strands.
• DNA polymerase an enzyme that synthesizes new
  DNA strands.
• Two PCR primers short DNA molecules
  (oligonucleotides) that define the DNA sequence
  to be amplified.
• Deoxynucleotide triphosphates (dNTPs) the
  building blocks for the newly synthesized DNA
  strands.
• Reaction buffer a chemical solution that
  provides the optimal environmental conditions.
• Magnesium a necessary cofactor for DNA
  polymerase activity.

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HPCR Amplify DNA?

• One PCR cycle consists of a DNA denaturation
  step, a primer annealing step and a primer
  extension step.
• DNA Denaturation Expose the DNA template to
  high temperatures to separate the two DNA strands
  and allow access by DNA polymerase and PCR
  primers.
• Primer Annealing Lower the temperature to allow
  primers to anneal to their complementary
  sequence.
• Primer E xtension Adjust the temperature for
  optimal thermostable DNA polymerase activity to
  extend primers.
• PCR uses a thermostable DNA polymerase so that
  the DNA polymerase is not heat-inactivated during
  the DNA denaturation step. Taq DNA polymerase is
  the most commonly used DNA polymerase for PCR.

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Mechanism of DNA Synthesis

• DNA polymerase extends the primer by sequentially
  adding a single dNTP (dATP, dGTP, dCTP or dTTP)
  that is complementary to the existing DNA strand
• The sequence of the newly synthesized strand is
  complementary to that of the template strand.
• The dNTP is added to the 3 end of the growing
  DNA strand, so DNA synthesis occurs in the 5 to
  3 direction.

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Instrumentation

• Thermal cyclers have a heat-conducting block to
  modulate reaction temperature.
• Thermal cyclers are programmed to maintain the
  appropriate temperature for the required length
  of time for each step of the PCR cycle.
• Reaction tubes are placed inside the thermal
  cycler, which heats and cools the heat block to
  achieve the necessary temperature.

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Thermal Cycling Programs

• A typical thermal cycler program is
• Initial DNA denaturation at 95C for 2 minutes
• 2035 PCR cycles
• Denaturation at 95C for 30 seconds to 1 minute
• Annealing at 4265C for 1 minute
• Extension at 6874C for 12 minutes
• Final extension at 6874C for 510 minutes
• Soak at 4C

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PCR Optimization

• Many PCR parameters might need to be optimized to
  increase yield, sensitivity of detection or
  amplification specificity. These parameters
  include
• Magnesium concentration
• Primer annealing temperature
• PCR primer design
• DNA quality
• DNA quantity

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Magnesium Concentration

• Magnesium concentration is often one of the most
  important factors to optimize when performing
  PCR.
• The optimal Mg2 concentration will depend upon
  the primers, template, DNA polymerase, dNTP
  concentration and other factors.
• Some reactions amplify equally well at a number
  of Mg2 concentrations, but some reactions only
  amplify well at a very specific Mg2
  concentration. 
• When using a set of PCR primers for the first
  time, titrate magnesium in 0.5 or 1.0mM
  increments to empirically determine the optimal
  Mg2 concentration.

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Primer Annealing Temperature

• PCR primers must anneal to the DNA template at
  the chosen annealing temperature.
• The optimal annealing temperature depends on the
  length and nucleotide composition of the PCR
  primers
• The optimal annealing temperature is often within
  5C of the melting temperature (Tm) of the PCR
  primer
• The melting temperature is defined as the
  temperature at which 50 of complementary DNA
  molecules will be annealed (i.e.,
  double-stranded).
• When performing multiplex PCR, where multiple DNA
  targets are amplified in a single PCR, all sets
  of PCR primers must have similar annealing
  temperatures.

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DNA Quality

• DNA should be intact and free of contaminants
  that inhibit amplification.
• Contaminants can be purified from the original
  DNA source.
• Heme from blood, humic acid from soil and melanin
  from hair
• Contaminants can be introduced during the
  purification process.
• Phenol, ethanol, sodium dodecyl sulfate (SDS) and
  other detergents, and salts.

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DNA Quantity

• DNA quantity
• More template is not necessarily better.
• Too much template can cause nonspecific
  amplification.
• Too little template will result in little or no
  PCR product.
• The optimal amount of template will depend on the
  size of the DNA molecule.

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Applications of PCR

• PCR and RT-PCR have hundreds of applications. In
  addition to targeting and amplifying a specific
  DNA or RNA sequence, some common uses include
• Labeling DNA or RNA molecules with tags, such as
  fluorophores or radioactive labels, for use as
  tools in other experiments.
• Cloning a DNA or RNA sequence
• Detecting DNA and RNA
• Quantifying DNA and RNA
• Genotyping and DNA-based identification

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Labeling DNA

• Labeling DNA with tags for use as tools (probes)
  to visualize complementary DNA or RNA molecules.
• Radioactive labels.
• Radioactively labeled probes will darken an X-ray
  film.
• Fluorescent labels (nonradioactive)
• Fluors will absorb light energy of a specific
  wavelength (the excitation wavelength) and emit
  light at a different wavelength (emission
  wavelength).
• The emitted light is detected by specialized
  instruments such as fluorometers.

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Quantitative PCR

• Avoids problems associated with the plateau
  effect, which reduces amplification efficiency
  and limits the amount of PCR product generated
  due to depletion of reactants, inactivation of
  DNA polymerase and accumulation of reaction
  products.
• The result of the plateau effect is that the
  amount of PCR product generated is no longer
  proportional to the amount of DNA starting
  material.
• The plateau effect becomes more pronounced at
  higher cycle numbers.
• Often performed in real time to monitor the
  accumulation of PCR product at each cycle.
• Real-time PCR allows scientists to quantify DNA
  before the plateau effect begins to limit PCR
  product synthesis.

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RT PCR
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THE PROBLEM

• NEED TO QUANTITATE DIFFERENCES IN mRNA EXPRESSION
• SMALL AMOUNTS OF mRNA
• LASER CAPTURE
• SMALL AMOUNTS OF TISSUE
• PRIMARY CELLS
• PRECIOUS REAGENTS

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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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NORTHERN
control
expt
10X
target gene
internal control gene actin, GAPDH, RPLP0 etc
2X
Corrected fold increase 10/2 5
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Standards

• same copy number in all cells
• expressed in all cells
• medium copy number advantageous
• correction more accurate

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Linear 20 to 1500
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Linear 20 to 1500
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REAL TIME PCR

• kinetic approach
• early stages
• while still linear

www.biorad.com
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SERIES OF 10-FOLD DILUTIONS
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SERIES OF 10-FOLD DILUTIONS
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SERIES OF 10-FOLD DILUTIONS
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threshold 300
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STANDARD CURVE METHOD
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PFAFFL METHOD

• M.W. Pfaffl, Nucleic Acids Research 2001
  292002-2007

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AFTER 1 CYCLE 100 2.00x 90 1.90x 80
1.80x 70 1.70x
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AFTER 1 CYCLE 100 2.00x 90 1.90x 80
1.80x 70 1.70x
AFTER N CYCLES fold increase (efficiency)n
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SERIES OF 10-FOLD DILUTIONS
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QUALITY CONTROL -EFFICIENCY CURVES

• use pcr baseline subtraction (not curve fitting
  default option) - see next slide
• set the threshold manually to lab standard
• check all melting curves are OK
• check slopes are parallel in log view
• delete samples if multiple dilutions cross line
  together (usually at dilute end of curve)
• delete samples if can detect amplification at
  cycle 10 or earlier
• make sure there are 5 or more points
• check correlation coefficient is more than 1.990

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Quality Control

• use pcr baseline subtraction (not curve fitting
  default option)
• set the threshold manually to lab standard
• check all melting curves are OK
• check slopes are parallel in log view
• delete samples if multiple dilutions cross line
  together (usually at dilute end of curve)
• delete samples if can detect amplification at
  cycle 10 or earlier
• make sure there are 5 or more points
• check correlation coefficient is more than 1.990

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tissue
extract RNA
copy into cDNA (reverse transciptase)
do real-time PCR
analyze results
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tissue
extract RNA
copy into cDNA (reverse transciptase)
do real-time PCR
analyze results
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• Should be free of protein (absorbance
  260nm/280nm)
• Should be undegraded (28S/18S 21)
• Should be free of DNA (DNAse treat)
• Should be free of PCR inhibitors
• Purification methods
• Clean-up methods

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OVERVIEW
tissue
extract RNA
copy into cDNA (reverse transciptase)
do real-time PCR
analyze results
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Importance of reverse transcriptase primers

• Oligo (dt)
• Random hexamer (NNNNNN)
• Specific

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Reverse Transcription

• adds a bias to the results
• efficiency usually not known

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tissue
extract RNA
copy into cDNA (reverse transciptase)
do real-time PCR
analyze results
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Importance of primers in PCR

• specific
• high efficiency
• no primer-dimers
• Ideally should not give a DNA signal
• cross exon/exon boundary

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Number Games
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Nested PCR
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Hot Start PCR
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Multiplex PCR
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RT PCR
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THANK U