Polymerase Chain Reaction

1
Polymerase Chain Reaction

• DNA amplification and much more

2
Table of Contents

• An introduction to PCR
• The PCR process
• What is PCR
• Typical components of a PCR
• PCR animation
• Typical PCR conditions
• PCR optimization
• Magnesium concentration
• Primer annealing temperature
• PCR primer design
• DNA quality and quantity

3
Table of Contents

• Applications of PCR
• Reverse transcription PCR
• DNA or RNA labeling
• DNA or RNA cloning
• DNA and RNA detection
• DNA and RNA quantitation
• Genotyping and DNA-based identification

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An Introduction to PCR

• To fully understand cellular processes
  scientists often examine events at the molecular
  level.
• Scientific studies often involve
• analysis of DNA RNA and protein molecules
• use of labeled DNA as a molecular tool to
  visualize these biomolecules
• Scientists need a quick and easy way to produce
  DNA in sufficient quantities for their studies
  and generate labeled DNA molecules to visualize
  and study specific molecules within cells.

5
What is PCR

• The polymerase chain reaction (PCR) is a
  relatively simple technique developed in 1985 to
  amplify sequence-specific DNA fragments in vitro.
• PCR is one of the most useful techniques in
  laboratories today due to its speed and
  sensitivity.
• Traditional techniques to amplify DNA require
  days or week. PCR can be performed in as little
  as 1 hour.
• Many biochemical analyses require the input of
  significant amounts of biological material PCR
  requires as little as one DNA molecule.
• These features make PCR extremely useful in basic
  research and commercial applications including
  genetic identity testing forensics industrial
  quality control and in vitro diagnostics.

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PCR Amplifies a Specific DNA Sequence

• PCR can be used to target a specific DNA
  subsequence in a much larger DNA sequence (e.g.
  a single 1000bp gene from the human genome which
  is 3 109bp).
• PCR allows exponential amplification of a DNA
  sequence.
• Each PCR cycle theoretically doubles the amount
  of DNA.
• During PCR an existing DNA molecule is used as a
  template to synthesize a new DNA strand.
• Through repeated rounds of DNA synthesis large
  quantities of DNA are produced.

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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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The PCR ProcessPCR Primers

• PCR primers
• Primers define the DNA sequence to be
  amplifiedthey give the PCR specificity.
• Primers bind (anneal) to the DNA template and act
  as starting points for the DNA polymerase since
  DNA polymerases can only extend existing DNA
  molecules and cannot initiate DNA synthesis
  without a primer.
• The distance between the two primers determines
  the length of the newly synthesized DNA
  molecules.

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How does PCR 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 Extension 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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PCR Animation

• View the PCR animation for a dynamic PCR
  demonstration.

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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

14
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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PCR Primer Design

• Many PCR failures can be avoided by designing
  good primers.
• Ideally all primers used in a PCR will have
  similar melting temperatures and GC content.
  Typically primers with melting temperatures in
  the range of 4570C are chosen. GC content
  should be near 50.
• Primers should have little intramolecular and
  intermolecular secondary structure which can
  interfere with primer annealing to the template.
• Primers with intramolecular complementarity can
  form secondary structure within the same primer
  molecule.
• Intermolecular complementarity allows a primer
  molecule to anneal to another primer molecule
  rather than the template.
• Software packages exist to design primers.

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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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Reverse Transcription PCR

• RNA can be amplified by including a simple
  reverse transcription (RT) step prior to PCR.
  This process is known as RT-PCR.
• Reverse transcriptases are RNA-dependent DNA
  polymerases which use an RNA template to make a
  DNA copy (cDNA). This cDNA can be amplified using
  PCR.

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RT-PCR Components

• Typical components of an RT-PCR include
• Reverse transcriptase the enzyme that
  synthesizes the cDNA copy of the RNA target.
• Reverse transcription primer a single short DNA
  molecule that acts as starting points for the
  reverse transcriptase since reverse
  transcriptases cannot initiate DNA synthesis
  without a primer.
• Deoxynucleotide triphosphates (dNTPs) the
  building blocks for the newly synthesized cDNA.
• Reaction buffer a chemical solution that
  provides the optimal environmental conditions.
• Magnesium a necessary cofactor for reverse
  transcriptase activity.
• All of the necessary PCR components for the PCR
  portion of RT-PCR.

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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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Identifying a DNA Sequence

• To study a specific DNA sequence that DNA
  sequence must be targeted and isolated (cloned)
  so that other surrounding DNA sequences do not
  interfere with the studies.
• PCR is faster and less labor-intensive than
  traditional cloning techniques.
• Traditional cloning techniques require weeks or
  months. PCR requires hours.
• PCR primers can be designed to amplify the exact
  sequence of interest.
• Traditional cloning techniques do not always
  yield the exact DNA fragment of interest.

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DNA and RNA Detection

• PCR can detect foreign DNA sequences in a
  biological sample.
• Example Hospitals often use PCR to detect
  bacteria and viruses and help diagnose illnesses.
• PCR can detect specific DNA sequences to
  characterize an organism.
• Example The multidrug resistance (MDR) gene
  confers resistance to antibiotics that are
  commonly used to treat bacterial infections. PCR
  using primers specific for the MDR gene will
  identify strains of bacteria that express MDR and
  are resistant to common antibiotics.
• RT-PCR can detect specific RNA sequences within a
  sample.
• Example Retroviruses have an RNA genome.
  Retroviral RNA can be detected by RT-PCR to
  diagnose retroviral infections.

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DNA and RNA Quantitation

• Quantitative PCR can be used to determine the
  copy number of a DNA sequence such as a gene
  within a genome or the number of organisms
  present in a sample (e.g. determining viral
  load).
• Quantitative RT-PCR is often used to quantitate
  the level of messenger RNA (mRNA) produced in a
  cell.
• As gene expression within a cell is activated or
  repressed the level of corresponding mRNA
  increases or decreases respectively.
• Quantitating mRNA levels by RT-PCR can tell us
  which genes are being up- or downregulated under
  certain conditions providing insight into gene
  function.

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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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Genotyping and DNA-Based Identification

• Cellular (genomic) DNA contains regions of
  variable sequences that differ between strains or
  even individual organisms.
• Variable regions are amplified by multiplex PCR
  and when the resulting DNA fragments are
  separated by size the resulting pattern acts
  like a unique barcode to identify a strain or
  individual.
• For human identification these variable regions
  often include short tandem repeats (STRs) and
  single-nucleotide polymorphisms (SNPs).
• STRs and SNPs are useful in DNA-based forensic
  investigations missing persons investigations
  and paternity disputes.

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DNA-Based Human Identification
The police collect a hair from a crime scene and
submit it for STR analysis (sample 1). Five
suspicious people were observed near the crime
scene shortly after the crime was committed. The
police collect DNA from these five people and
submit it for STR analysis (samples 26). Do any
of these five DNA samples match the DNA from the
hair collected at the cime scene