Polymerase Chain reaction(PCR)

Narayan Ghimire
M.Sc. Medical Biochemistry
2nd semester
SHAS,PU
Polymerase Chain Reaction(PCR)
Amplification techniques of DNA
 Introduction of PCR
• PCR is a means to amplify a particular piece of DNA .
• Amplify= making numerous copies of a segment of DNA.
• PCR can make billions of copies of a target sequence of DNA in short
time.
• It is a laboratory version of DNA Replication in cells.
• The laboratory version is commonly called “in vitro” since it occurs in a
test tube while “in vivo” signifies occurring in a living cell
 History
• 1966, Thomas Brock discovers Thermus Aquaticus, a thermostable
bacteria in the hot springs of Yellowstone National Park
• 1983, Kary Mullis postulated the concept of PCR ( Nobel Prize in 1993)
• 1985, Saiki publishes the first application of PCR ( beta-Globin)
• 1985, Cetus Corp. Scientists isolate Thermostable Taq Polymerase (from
T.Aquaticus), which revolutionized PCR
 Basic Principle
• The PCR technique is based on the enzymatic replication of DNA.

• In PCR, a short segment of DNA is amplified using primer mediated

enzymes.

• DNA Polymerase synthesises new strands of DNA complementary to the

template DNA.

• The DNA polymerase can add a nucleotide to the pre-existing 3’-OH group
only. Therefore, a primer is required. Thus, more nucleotides are added to
the 3’ prime end of the DNA polymerase.
 PCR Component
• Water
• Buffer
• DNA template
• Primers
• Nucleotides
• Mg++ ions
• DNA
Polymerase
 Water
– The medium for all other components.

 Buffer
– Stabilizes the DNA polymerase, DNA, and nucleotides
– 500 mM KCl
– 100 mM Tris-HCl, pH 8.3
– Triton X-100 or Tween

 DNA template
– Contains region to be amplified
– Any DNA desired
– Purity not required
– Should be free of polymerase inhibitors
 Primers
– Specific for ends of amplified region
– Forward and Reverse
– Annealing temperatures should be
known
• Depends on primer length, GC
content, etc.
– Length 15-30 nt
– Conc 0.1 – 1.0 uM (pMol/ul)
 Nucleotides
• Standard PCRs contain equimolar concentrations of (200-250 μM each).

 dCTP, Deoxycytidine triphosphated, dTTP, Deoxythymidine

triphosphate, dATP, Deoxyadenosine triphosphated, dGTP,
Deoxyguanosine triphosphate

• These dNTPs are available commercially and supplied as pyrophosphate

free mixtures.

• dNTPs must be stored at -20 ºC.

•
• During long term storage, small amounts of water evaporate and freeze
over the walls of the vial.

• Hence in order to minimize the change in concentration, vials should be

centrifuged before use.
Free Divalent cations:
– Essential co-factor of DNA polymerase
– Usually Mg2+ used .
– Too little: Enzyme won’t work.
– Stabilizes the DNA double-helix
– Too much: DNA extra stable, non-specific priming, band smearing
– Used at 0.5 to 3.5 uM in the assay
 DNA Polymerase
• A thermostable DNA polymerase, which can withstand the denaturation
temperatures (94-95 ºC), is essential to catalyze the template dependent
synthesis of DNA.

• Originally the Klenow fragment of Escherichia coli pol I was used which was
inactivated at high temperatures required for separation of two strands of DNA
and hence had to be added freshly after every denaturation step.

• This hurdle was overcome with the introduction of thermostable DNA

polymerase, Taq DNA polymerase isolated from thermophilic bacterium
Thermus aquatics.

• For a standard 25-50μl reaction, 0.5-0.25 units of Taq polymerase are used
Controls

1) Reagent control/no-template control : Consists of all PCR reagents except

DNA template to ensure no contamination of PCR reaction.

2) Negative control : Typically cell line that is negative for the target and
used to ensure specificity of the PCR reaction.

3) Positive control : Undiluted or diluted cell lines to ensure precision and

prevent run-to-run variation in quantification. A sensitivity control may
be included to ensure detection of low-level targets.

Others

• Some researchers have reported that the efficiency of reaction is increased

by inclusion of 10% dimethyl sulfoxide (DMSO) in the Taq polymerase
buffer
GC content:
• GC% = Number of G's and C's in the primer as a percentage of the total
bases.
• Should be 40-60%.
GC clamp:
• Presence of G or C bases within the last five bases from the 3' end of primers.
• Not more than 2 G's or C's .

Melting temperature (Tm):

• Melting temperatures in the range of 50-60 °C generally produce the best
results.
• Maximum difference between primer pairs is 5°C.
• The Tm of the primer can be calculated by the following formula:

• Tm = [(G + C) x 4] + [(A + T) x 2]
 Annealing Temperature (Ta):
• The primer melting temperature is the estimate of the DNA-DNA hybrid
stability and critical in determining the annealing temperature.
• Depends directly on length and GC composition of the primers.
• Too high Ta : produce insufficient primer-template hybridization.
• Too low Ta : lead to non-specific products caused by a high number of
base pair mismatches.
Stages
Temperature cycling
 Procedure
• Amplification can be conveniently performed in a DNA thermal cycler.
• The reaction mixture contains
 A target sequence of 100-500 base pair length

 50 mM KCl

 10 mM Tris.HCI (pH of 8.4 at room temp)

 1.5 mM MgCl2

 100 μg/ml Gelatine

 0.25 μM of each Primer

 200 μM of each Deoxynucleotide Triphoshates (dATP, dCTP, dGTP, & dTTP)

 2.5’ units of Taq Polymerase

 Cycling parameter
1. Initial Denaturation 95 C 3 min
2. DNA Denaturation 95 C 1 min
3. Primer Annealing 65 C 1 min
4. Primer Extension 72 C 1 min
5. Go to step #2, repeat 39 more times
6. End
• Amplification of the target sequence is achieved by a repetitive series of
cycles involving three steps:
1. Denaturation of the template by heat.
2. Annealing of the oligonucleotide primers to single stranded target
sequences.
3. Extension of the annealed primers by thermostable DNA polymerase.
 1. Denaturation
• In PCRs catalyzed by Taq polymerase, denaturation is carried out at 94-95
ºC, which is the highest temperature the enzyme can withstand for 30 or
more cycles without being damaged.

• During the first cycle, denaturation is carried out for 5 minutes to ensure
complete denaturation of the long molecules of template DNA.
•
• But at times such longer duration of denaturation may be deleterious.

• Denaturation for 45 seconds at a temperature of 94-95ºC is recommended

for routine amplification of linear DNA templates containing 55% or lesser
amount of G+C.
• Higher temperatures may be required to denaturate templates containing
higher amounts of G+C.

• In addition, longer the DNA templates, greater is the denaturation time

required.

• If denaturation temperature is too low or if time is too short, only the A–T
rich regions of template DNA will be denatured.

• Such DNA will re-anneal back when denaturation temperature is reduced

later during PCR cycle
 2. Annealing
• Annealing temperatures range from 55-65 ºC depending on the primer
sequence and length.

• Annealing temperature is critical.

 If annealing temperature is too high: the oligonucleotide primers anneal
poorly and the amplified DNA is too low.
 If annealing temperature is too low: nonspecific annealing of primers may
occur, resulting in the amplification of unwanted segments of DNA.

• Annealing temperature can be optimized by performing series of trial

PCRs at room temperatures ranging from 2-100 ºC below the melting
temperatures of oligonucleotide primers. Also a sequential series of
annealing temperatures can be used in a routine PCR
 3. Extension
• DNA polymerase catalyzes the extension of oligonucleotide primer
resulting in synthesis of a new strand, having sequences complementary to
template strand.

• Optimal temperature for DNA synthesis may vary slightly depending on

the DNA polymerase used.
•
• When Taq polymerase is used, the ideal temperature for DNA synthesis is
about 72-78 ºC.

• Taq polymerase can insert about 2000 nucleotides every minute at this
temperature.
• Number of cycles needed for amplification depends on:
 Number of template DNA sequences present in the reaction mixture
 Efficiency of primer extension
 PCR phases
• A basic PCR run can be broken up into three phases:

1. EXPONENTIAL
• Exact doubling of product which accumulate at every cycle (assuming
100% reaction efficiency).
• Reaction is very specific and precise.

2. LINEAR (HIGH VARIABILITY):

• Reaction components are being consumed, the reaction is slowing, and
products are starting to degrade.
3. PLATEAU (END-POINT)
• Reaction has stopped, no more products are being made and if left long
enough, the PCR products will begin to degrade.

• In most cases the plateau is unavoidable but by the time it occurs, adequate
amounts of product will have accumulated.

• If more material is required, multiple reactions can be easily set up.

 Visualization Method Of PCR amplified
Products
• After thermal cycling, tubes are taken out of the PCR machine.
• Contents of tubes are loaded onto an agarose gel.
• DNA is separated by size using an electric field.
• DNA is then stained.
• PCR products are visible as different “bands”.
 Application of PCR
• Genotyping.
 RT-PCR.
 Cloning.
 Mutation detection.
 Sequencing.
 Microarrays.
 Forensics.
 Paternity testing
Advantages and Limitation of PCR
 Modification of PCR
• In recent years, modifications or variants have been developed from the
basic PCR method to improve performance and specificity, and to achieve
the amplification of other molecules of interest in research as RNA.

i. Multiplex PCR which simultaneously amplify several DNA sequences

(usually exonic sequences).

ii. Nested PCR increases the specificity of the amplified product for a
second PCR with new primers that hybridize within the amplified fragment in
the first PCR.

iii. Semiquantitative PCR which allows an approximation to the relative

amount of nucleic acids present in a sample.

iv. RT-PCR which generates amplification of RNA by synthesis of cDNA

(DNA complementary to RNA) that is then amplified by PCR
 Quantitative PCR(qPCR)
• One of the most useful PCR applications is quantitative PCR or qPCR.

• used to measure the specific amount of target DNA ( cDNA/RNA) in

sample.

• By measuring amplification only within the phase of true exponential

increase,the amount of measured product more accurately reflects the
initial amount of target.

• Special thermal cyclers are used that monitor the amount of product
during the amplification.
• In its simplest form a PCR is set up that includes a DNA-binding cyanine
dye such as SYBR green.

• This dye binds to the major groove of double-stranded DNA but not
single-stranded DNA and so as amplicons accumulate during the PCR
process SYBR green binds the double-stranded DNA proportionally and
fluorescence emission of the dye can be detected following excitation.

• Thus the accumulation of DNA amplicons can be followed in real time

during the reaction run.
SYBR green
 The TaqMan system

• In order to make qPCR specific a number of strategies may be employed

that rely on specific hybridization probes.

• One ingenious method is called the TaqMan assay or 5 ꞌnuclease assay.

• Here the probe consists of an oligonucleotide labelled with a fluorescent

reporter at one end of the molecule and quencher at the other end.

• The PCR proceeds as normal and the oligonucleotide probe binds to the
target sequence in the annealing step.
• As the Taq polymerase extends from the primer its 5ꞌ exonuclease activity
degrades the hybridization probe and releases the reporter from the
quencher.

• A signal is thus generated which increases in direct proportion to the

number of starting molecules and fluorescence can be detected in real time
as the PCR proceed.

• Although relatively expensive in comparison to other methods for

determining expression levels.

• It is simple, rapid and reliable and now in use in many research and clinical
areas
 Real Time - PCR
 This same principle of amplification of PCR is employed in real-time PCR.

 But instead of looking at bands on a gel at the end of the reaction, the
process is monitored in “real-time”.

 The reaction is placed into a real-time PCR machine that watches the
reaction occur with a camera or detector.

 Although many different techniques are used to monitor the progress of a

PCR reaction, all have one thing in common. They all link the amplification
of DNA to the generation of fluorescence which can simply be detected with
a camera during each PCR cycle. Hence, as the number of gene copies
increases during the reaction, so does the fluorescence, indicating the
progress of the reaction.
 Components of Real Time PCR
Real-time PCR instruments consist of THREE main components:

1. Thermal Cycler (PCR machine)

2. Optical Module (to detect fluorescence in the tubes during the run)

3. Computer (to translate the fluorescence data into meaningful

results)
Software interface
 Cycle of Threshold
We describe the position of the lines with a value that represents the
cycle number where the trace crosses an arbitrary threshold.
This is called the “Ct Value”.
Ct values are directly related to the starting quantity of DNA, by way of
the formula:

Quantity = 2^Ct
Reverse Transcriptase-PCR/RT-PCR
• Used to generate cDNA copies from extracted mRNAs.
• Types : One step RT-PCR and Two step RT-PCR

• This reaction is catalyzed by retrovirus reverse transcriptase (reverse

transcriptase) which synthesizes a DNA chain from an RNA template.

• At first, the total RNAs are extracted & the mRNAs are isolated from the
total RNA by affinity chromatography using oligodT(polyT oligonucleotide).

• Then, the mRNAs are subjected to reverse transcriptase which will

generate a copy of DNA (cDNA) of each mRNA.
• After the reverse transcription, the mRNAs are hydrolyzed (alkaline
treatment, RNase, or temperature).

• The following steps are carried out in the enclosure of the thermal cycler.

• The single-stranded cDNAs are then replicated by the DNA polymerase

during a first temperature cycle.

• Other cycles are repeated to amplify double-stranded cDNAs in large

quantities.

• The analysis of the expression variations of genes involved in a pathology

can lead to new therapeutic or diagnostic targets.
 Based on process of reverse transcription , which reverse transcribes the
RNA into DNA and was initially isolated from retroviruses.

 First step of RT-PCR “first strand reaction ‘’- synthesis of cDNA using
oligonucleotide dT primers (37 degree Celsius for 1 hour.

 “Second strand reaction “- Digestion of cDNA:RNA hybrid (RNAseH).

 Allows the detection of even rare or low copy mRNA sequences by

amplifying its complementary DNA
 Multiplex PCR
• Widespread molecular biology technique.

• Amplification of single template as well as multiple templates in a single

PCR experiment.

• By using mulltiple primer pairs in a reaction mixture.

• Types : single template PCR reaction and multiple template PCR reaction
Advantage :

• It has the potential to produce considerable saving in time and effort

within the laboratory.

• Without compromising on the utility of the experiment.

• Disadvantages

• Optimization is difficult;since many sets of forward and reverse primers

are to be designed for use.

• Increase cost.

• Presence of multiple primer may lead to cross hybridization with each

other and the possibility of mis-priming wth other templates.
 Nested PCR
• Two pairs of primers instead one pair are used to amplify a fragment.

• In the first PCR, one pair of primers is used to generate DNA

products,which may contain products amplified from non target areas.

• Template ( product of first PCR) in a second PCR,using one(Hemi-nesting)

or two different primers whose binding sites are located(nested) within
the first set , thus increasing specificity.

• First round PCR : copies the larger regions

• Second round PCR : copies Target region
Nested PCR
 Inverse PCR
• Inverse PCR (Ochman et al.,1988) uses standard PCR- Primers oriented in reverse
direction of the usual orientation.

• Also called as inverted PCR/ Inside out PCR

• The template for reverse primers is a restriction fragment that has been
selfligated.

• Inverse PCR functions to clone sequences flanking a known sequence. Flanking

DNA sequences are digested and then ligated to generate circular DNA.

APPLICATION

• Amplification and identification of Flanking sequence such as transposable

element, and identification of genomic inserts .
Inverse PCR
 Hot start PCR
• This is a technique that reduces non specific amplification during the initial
set up stages of the PCR by inactivating the Taq polymerase at lower room
temperature.

• This technique may be performed manually by heating the reaction

components to the melting temperature(e.g. 95 degree celcius) before
adding the polymerase.

• DNA polymerase: Eubacterial type I DNA polymerase,Pfu

• These thermophilic DNA polymerase show a very small polymerase activity

at room temperature.

Types : Mechanical Hot start PCR and Non – Mechanical Hot Start PCR
 Touchdown PCR
• In this type,the annealing temperature is gradually decreased in later cycles.

• The annealing temperature in the early first 2 cycles is usually 3-5 ( but upto
10) degree celcius above the standard Tm of the primers used,while in the
later cycles it is a similar amount below the Tm( reduced by 1 degree celcius
for every 1 or 2 cycles).

• When annealing temperature reaches at an optimum point ,then specific

amplification of the target DNA starts.

• The initial higher annealing temperature leads to greater specificity for

primer binding,while the lower temperatures permit more efficient
amplification at the end of the reaction
Touchdown PCR
 Application of Touchdown PCR
• It is used to optimize yield of amplified product at different annealing
temperatures.

• In some cases it is very tricky to amplify DNA of mismatches between

oligonucleotide primers and the template DNA and it is not possible to
calculate annealing temperature.
Troubleshooting in PCR
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