Transcript POLYMERASE CHAIN REACTION - Universitas Sebelas Maret

POLYMERASE CHAIN
REACTION
PCR
Amplification of a specific DNA sequence (1005000 bp)
 2 synthetic oligonucleotide primers flanking the
target sequence
 Use of thermostable DNA polymerase
 3 step cycling process :
-denaturation
Repeated cycle
-annealing of primers
-extension
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history
Kary Mullis
 1st thermal cycler :1986
 3 waterbaths with different temperature,
non thermostable DNA polymerase
 Polymerase from Thermus aquaticus (Taq
polymerase) : Topt:72C,withstand heating
to 100C
 Other polymerase: Pfu
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DENATURATION
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Denaturation: heating DNA to temperature above the Tm to make
ssDNA.
if the DNA is heated in buffers of ionic strength lower than 150mM
NaCl, the melting temperature is generally less than 100oC - which
is why PCR works with denaturing temperatures of 91-97oC.
Taq polymerase has a half-life of 30 min at 95oC, one should
not do more than about 30 amplification cycles: however, it is
possible to reduce the denaturation temperature after about 10
rounds of amplification, as the mean length of target DNA is
decreased: for templates of 300bp or less, denaturation
temperature may be reduced to as low as 88oC for 50%
(G+C) templates (Yap and McGee, 1991), which means one may do
as many as 40 cycles without much decrease in enzyme efficiency.
DENATURATION
"Time at temperature" is the main reason for
denaturation / loss of activity of Taq: thus, if one
reduces this, one will increase the number of
cycles that are possible, whether the
temperature is reduced or not.
 Normally the denaturation time is 1 min at
94oC: it is possible, for short template
sequences, to reduce this to 30 sec or less.
 Increase in denaturation temperature and
decrease in time may also work: Innis and
Gelfand (1990) recommend 96oC for 15 sec.
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annealing
T melting : depend on Primer length and
sequence, the melting temperature of a DNA duplex
increases with its length and (G+C) content
 a simple formula for calculation of the primers Tm is
Tm = 4(G + C) + 2(A + T)oC
 annealing temperature (Ta) about 5oC below the
lowest Tm of the pair of primers to be used (Innis
and Gelfand, 1990)
 if the Ta is increased by 1oC every other cycle,
specificity of amplification and yield of products
<1kb in length are both increased
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annealing
If a Ta is too low : one or both primers will
anneal to sequences other than the true
target, as internal single-base mismatches or
partial annealing may be tolerated
 this is fine if one wishes to amplify similar
or related targets; however, it can lead
to "non-specific" amplification and
consequent reduction in yield of the desired
product
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annealing
too high Ta : too little product will be made,
as the likelihood of primer annealing is reduced
 a pair of primers with very different Tas may
never give appreciable yields of a unique
product, and may also result in inadvertent
"asymmetric" or single-strand amplification of
the most efficiently primed product strand.
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Annealing does not take long: most primers
will anneal efficiently in 30 sec or less, unless the Ta
is too close to the Tm, or unless they are unusually
long
An illustration of the effect of annealing temperature on the specificity and on the
yield of amplification of Human papillomavirus type 16 (HPV-16)
(Williamson and Rybicki, 1991: J Med Virol 33: 165-171)
Plasmid and biopsy sample DNA templates were amplified at different
annealing temperatures as shown: note that while plasmid is amplified
from 37 to 55oC, HPV DNA is only specifically amplified at 50oC.
EXTENSION
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normally 70 - 72oC, for 0.5 - 3 min
elongation occurs from the moment of
annealing, at around 70oC the activity is optimal, and
primer extension occurs at up to 100 bases/sec
About 1 min is sufficient for reliable amplification
of 2kb sequences (Innis and Gelfand, 1990). Longer
products require longer times: 3 min is a good bet for
3kb and longer products. Longer times may also be
helpful in later cycles when product concentration
exceeds enzyme concentration (>1nM), and when dNTP
and / or primer depletion may become limiting.
REACTION BUFFER
Recommended buffers generally contain :
 10-50mM Tris-HCl pH 8.3,
 up to 50mM KCl, 1.5mM or higher MgCl2,
 primers 0.2 - 1uM each primer,
 50 - 200uM each dNTP,
 gelatin or BSA to 100ug/ml,
 and/or non-ionic detergents such
as Tween-20 or Nonidet P-40 or Triton X100 (0.05 - 0.10% v/v)
(Innis and Gelfand, 1990). Modern formulations
may differ considerably
REACTION BUFFER
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Higher than 50mM KCl or NaCl inhibits Taq, but
some is necessary to facilitate primer annealing
[Mg2+] affects primer annealing; Tm of template,
product and primer-template associations; product
specificity; enzyme activity and fidelity. Taq
requires free Mg2+, so allowances should be made for
dNTPs, primers and template, all of which chelate and
sequester the cation; of these, dNTPs are the most
concentrated, so [Mg2+] should be 0.5 2.5mM greater than [dNTP]. A titration should be
performed with varying [Mg2+] with all new
template-primer combinations, as these can differ
markedly in their requirements, even under the same
conditions of concentrations and cycling
times/temperatures.
REACTION BUFFER
Some enzymes do not need added
protein, others are dependent on it. Some
enzymes work markedly better in the presence
of detergent, probably because it prevents the
natural tendency of the enzyme to aggregate.
 Primer concentrations should not go above
1uM unless there is a high degree of
degeneracy; 0.2uM is sufficient for homologous
primers.
 Nucleotide concentration need not be above
50uM each: long products may require more,
however.
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Cycle Number
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The number of amplification cycles necessary to produce a
band visible on a gel depends largely on the starting
concentration of the target DNA
Innis and Gelfand (1990) recommend from 40 - 45 cycles to
amplify 50 target molecules, and 25 - 30 to amplify 3x105
molecules to the same concentration.
This non-proportionality is due to a so-called plateau effect, which
is the attenuation in the exponential rate of product accumulation in
late stages of a PCR, when product reaches 0.3 - 1.0 nM. This may
be caused by degradation of reactants (dNTPs, enzyme); reactant
depletion (primers, dNTPs - former a problem with short products,
latter for long products); end-product inhibition (pyrophosphate
formation); competition for reactants by non-specific products;
competition for primer binding by re-annealing of concentrated
(10nM) product (Innis and Gelfand, 1990).
If desired product is not made in 30 cycles, take a small sample (1ul) of
the amplified mix and re-amplify 20-30x in a new reaction mix rather than
extending the run to more cycles: in some cases where template
concentration is limiting, this can give good product where extension of
cycling to 40x or more does not
nested primer PCR
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PCR amplification is performed with one
set of primers, then some product is taken
- with or without removal of reagents - for
re-amplification with an internally-situated,
"nested" set of primers. This process
adds another level of specificity, meaning
that all products non-specifically amplified
in the first round will not be amplified in
the second
This gel photo shows the effect of nested PCR amplification on the
detectability of Chicken anaemia virus (CAV) DNA in a dilution series: the
PCR1 just detects 1000 template molecules; PCR2 amplifies 1 template
molecule (Soiné C, Watson SK, Rybicki EP, Lucio B, Nordgren RM, Parrish
CR, Schat KA (1993) Avian Dis 37: 467-476).
Helix Destabilisers / Additives
With NAs of high (G+C) content, it may be necessary to use
harsher denaturation conditions. For example, one may
incorporate up to 10% (w or v/v) :
 dimethyl sulphoxide (DMSO),
 dimethyl formamide (DMF),
 urea
 or formamide
These additives are presumed to lower the Tm of the target NA,
although DMSO at 10% and higher is known to decrease the activity of
Taq by up to 50% (Innis and Gelfand, 1990; Gelfand and White, 1990).
Additives may also be necessary in the amplification of long target
sequences: DMSO often helps in amplifying products of
>1kb. Formamide can apparently dramatically improve the specificity of
PCR (Sarkar et al., 1990), while glycerol improves the amplification of
high (G+C) templates (Smith et al., 1990)
Polyethylene glycol (PEG) may be a useful additive when DNA template
concentration is very low: it promotes macromolecular association by solvent
exclusion, meaning the pol can find the DNA
A simple set of rules for primer sequence design (adapted from
Innis and Gelfand, 1991):
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primers should be 17-28 bases in length;
base composition should be 50-60% (G+C);
primers should end (3') in a G or C, or CG or GC: this prevents
"breathing" of ends and increases efficiency of priming;
Tms between 55-80oC are preferred;
runs of three or more Cs or Gs at the 3'-ends of primers may
promote mispriming at G or C-rich sequences (because of stability
of annealing), and should be avoided;
3'-ends of primers should not be complementary (ie. base pair),
as otherwise primer dimers will be synthesised preferentially to
any other product;
primer self-complementarity (ability to form SECONDARY
structures such as hairpins) should be avoided.
PRIMER DESIGN
Requires knowledge of some sequence
information
 Gives high specificity and sensitivity
 Allows amplification from limited starting
material
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APPLICATION OF PCR
DIAGNOSTIC
 BIODIVERSITY ANALYSIS
 LABORATORY ROUTINE
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Diagnostic Applications of PCR
detecting pathogens using genomespecific primer pairs
 screening specific genes for unknown
mutations
 genotyping using known STS (Sequence
Tagged Sites) markers
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Laboratory applications of PCR
subcloning DNA targets using PCR,
-T/A Cloning
-Restriction Site Addition
-Blunt-end Ligation
 PCR-mediated in vitro mutagenesis.
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Reverse transcription
polymerase chain reaction
Amplification from mRNA template
 RNA strand is first reverse transcribed into its DNA complement
(complementary DNA, or cDNA) using the enzyme reverse transcriptase,
and the resulting cDNA is amplified using traditional PCR
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The two-step RT-PCR process for converting RNA to DNA and the
subsequent PCR amplification of the reversely-transcribed DNA:
First strand reaction: complementary DNA (cDNA) is made from an mRNA
template using dNTPs & reverse transcriptase. The components are
combined with a DNA primer in a reverse transcriptase buffer for an
hour at 42°C.
Second strand reaction: after the reverse transcriptase reaction is
complete, cDNA has been generated from the original ss mRNA,
standard PCR (called the “second strand reaction”) is initiated.
In the two-step RT-PCR a thermostable DNA polymerase & the
upstream and downstream DNA primers are added. Heating the
reaction to temperatures above 37°C facilitates binding of DNA
primers to the cDNA, & subsequent higher temperatures allow the
DNA polymerase to make double-stranded DNA from the cDNA.
Heating the reaction to ~95°C melts the two DNA strands apart,
enabling the primers to bind again at lower temperatures and begin
the chain reaction again. After ~30 cycles, millions of copies of the
sequence of interest are generated
The use of RT-PCR
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the diagnosis of genetic diseases
the determination of the abundance of specific different
RNA molecules within a cell or tissue as a measure
of gene expression
the cloning of eukaryotic genes in prokaryotes
most eukaryotic genes contain introns which are present
in the genome but not in the mature mRNA, the cDNA
generated from a RT-PCR reaction is the exact DNA
sequence which would be directly translated into protein
after transcription. When these genes are expressed in
prokaryotic cells such as E. coli for protein
production/purification, the RNA produced directly from
transcription need not undergo splicing as the transcript
contains only exons (prokaryotes lack the mRNA splicing
mechanism of eukaryotes)
studying the genomes of viruses whose genomes are
composed of RNA, such as retroviruses like HIV
Real-time polymerase chain reaction
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amplify and simultaneously quantify a
targeted DNA molecule. measurement of DNA
amplification during PCR in real time, i.e., the amplified
product is measured at each PCR cycle
Two common methods of quantification are: (1) the use
of fluorescent dyes that intercalate with double-stranded
DNA, and (2) modified DNAoligonucleotide probes
that fluoresce when hybridized with a complementary
DNA
Frequently, real-time polymerase chain reaction is
combined with reverse transcription to
quantify messenger RNA (mRNA) in cells or tissues
Real-time PCR using doublestranded DNA dyes
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A DNA-binding dye binds to all dsDNA in PCR, causing fluorescence
of the dye. An increase in DNA product during PCR therefore leads
to an increase in fluorescence intensity and is measured at each
cycle, thus allowing DNA concentrations to be quantified. However,
dsDNA dyes such as SYBR Green will bind to all dsDNA PCR
products, including nonspecific PCR products (such as
"primer dimers"). This can potentially interfere with or prevent
accurate quantification of the intended target sequence
The reaction is run in a thermocycler, and after each cycle, the
levels of fluorescence are measured with a detector; the dye only
fluoresces when bound to the dsDNA (i.e., the PCR product). With
reference to a standard dilution, the dsDNA concentration in the
PCR can be determined
SYBR Green can’t bind single stranded DNA or primer
But it can bind double stranded DNA once the polymerase has made the 2nd strand
Fluorescent reporter probe method
uses a sequence-specific RNA or DNA-based
probe to quantify only the DNA containing the
probe sequence; therefore, use of the reporter
probe significantly increases specificity
 allows quantification even in the presence of
some non-specific DNA amplification
 allows for multiplexing - assaying for several
genes in the same reaction by using specific
probes with different-coloured labels, provided
that all genes are amplified with similar
efficiency
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commonly carried out with an RNA-based probe with a
fluorescent reporter at one end and a quencher of
fluorescence at the opposite end of the probe. The close
proximity of the reporter to the quencher prevents
detection of its fluorescence; breakdown of the probe by
the 5' to 3' exonuclease activity of the taq
polymerase breaks the reporter-quencher proximity and
thus allows unquenched emission of fluorescence, which
can be detected. An increase in the product targeted by
the reporter probe at each PCR cycle therefore causes a
proportional increase in fluorescence due to the
breakdown of the probe and release of the reporter
The PCR is prepared as usual and the reporter probe
is added.
As the reaction commences, during
the annealing stage of the PCR both probe and
primers anneal to the DNA target.
Polymerisation of a new DNA strand is initiated from
the primers, and once the polymerase reaches the
probe, its 5'-3-exonuclease degrades the probe,
physically separating the fluorescent reporter from
the quencher, resulting in an increase in
fluorescence.
Fluorescence is detected and measured in the realtime PCR thermocycler, and its geometric increase
corresponding to exponential increase of the
product is used to determine the threshold cycle
(CT) in each reaction.
(1) In intact probes, reporter fluorescence is quenched.
(2) Probes and the complementary DNA strand are
hybridized and reporter fluorescence is still
quenched.
(3) During PCR, the probe is degraded by the Taq
polymerase and the fluorescent reporter released.
Principle of quantitation
The number of cycles it takes to reach a certain amount of fluorescence is
proportional to the amount of cDNA present at the start
 This can be plotted as cycle no v log concentration to give a straight line
 By using known amounts of cDNA you can obtain a standard curve to find
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out levels of an unknown
The use of real time PCR
rapidly detect the presence of genes involved
in infectious diseases, cancer and genetic
abnormalities
 determining how the genetic expression of a
particular gene changes over time, such as in
the response of tissue and cell cultures to an
administration of a pharmacological agent,
progression of cell differentiation, or in response
to changes in environmental conditions
 used in environmental microbiology, for example
to quantify resistance genes in water samples
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