Polymerase Chain Reaction
DNA amplification
and much more
Table of Contents
• An introduction to PCR
• The PCR process
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What is PCR?
Typical components of a PCR
PCR animation
Typical PCR conditions
• PCR optimization
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Magnesium concentration
Primer annealing temperature
PCR primer design
DNA quality and quantity
Table of Contents
• Applications of PCR
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Reverse transcription PCR
DNA or RNA labeling
DNA or RNA cloning
DNA and RNA detection
DNA and RNA quantitation
Genotyping and DNA-based identification
An Introduction to PCR
• To fully understand cellular processes, scientists often
examine events at the molecular level.
• Scientific studies often involve:
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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.
What is PCR?
• The polymerase chain reaction (PCR) is a relatively
simple technique developed in 1985 to amplify sequencespecific DNA fragments in vitro.
• PCR is one of the most useful techniques in laboratories
today due to its speed and sensitivity.
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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.
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.
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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.
The PCR Process—Reaction Components
Typical components of a PCR include:
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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.
The PCR Process—PCR Primers
PCR primers
• Primers define the DNA sequence to be amplified—they
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.
How does PCR Amplify DNA?
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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.
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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.
PCR Animation
View the PCR animation for a dynamic PCR
demonstration.
Mechanism of DNA Synthesis
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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.
Instrumentation
• Thermal cyclers have a heat-conducting block to
modulate reaction temperature.
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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.
Thermal Cycling Programs
A typical thermal cycler program is:
Initial DNA denaturation at 95°C for 2 minutes
20–35 PCR cycles: Denaturation at 95°C for 30 seconds to 1 minute
Annealing at 42–65°C for 1 minute
Extension at 68–74°C for 1–2 minutes
Final extension at 68–74°C for 5–10 minutes
Soak at 4°C
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
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.
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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.
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 5°C 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.
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 45–70°C 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.
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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.
DNA Quality
• DNA should be intact and free of contaminants that inhibit
amplification.
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Contaminants can be purified from the original DNA source.
• Heme from blood, humic acid from soil and melanin from hair
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Contaminants can be introduced during the purification process.
• Phenol, ethanol, sodium dodecyl sulfate (SDS) and other detergents,
and salts.
DNA Quantity
DNA quantity
• More template is not necessarily better.
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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.
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.
DNA
RNA
Reverse
transcription
cDNA
PCR
DNA DNA DNA
DNA DNA DNA
DNA
DNA
RT-PCR Components
Typical components of an RT-PCR include:
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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.
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
Labeling DNA
• Labeling DNA with tags for use as tools (probes) to
visualize complementary DNA or RNA molecules.
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Radioactive labels.
• Radioactively labeled probes will darken an X-ray film.
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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.
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.
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Traditional cloning techniques require weeks or months. PCR
requires hours.
• PCR primers can be designed to amplify the exact
sequence of interest.
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Traditional cloning techniques do not always yield the exact DNA
fragment of interest.
DNA and RNA Detection
• PCR can detect foreign DNA sequences in a biological
sample.
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Example: Hospitals often use PCR to detect bacteria and viruses
and help diagnose illnesses.
• PCR can detect specific DNA sequences to characterize
an organism.
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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.
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Example: Retroviruses have an RNA genome. Retroviral RNA can
be detected by RT-PCR to diagnose retroviral infections.
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.
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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.
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.
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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.
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Real-time PCR allows scientists to quantify DNA before the plateau
effect begins to limit PCR product synthesis.
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.
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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.
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 #2–6).
Do any of these five DNA
samples match the DNA from
the hair collected at the cime
scene?