TYPES OF POLYMERASE CHAIN
REACTION (PCR)
M.Abdul Sattar
Types
Hot-startPCR
Colony PCR
Nested PCR
Tochdown PCR
Multiplex PCR
RT –PCR
Real Time PCR
Hot-start PCR
This technique reduces non-specific
amplification during the initial set up stages
of the PCR.
It may be performed manually by heating the
reaction components to the melting
temperature (e.g., 95˚C) before adding the
polymerase.
In conventional PCR, the Taq DNA
polymerase is active at room temperature
and to a lesser degree, even on ice. In some
instances, when all the reaction components
are put together, nonspecific primer
annealing can occur due to these low
temperatures.
This nonspecific annealed primer can
generating nonspecific products and
lowering product yields.
The hot start PCR is a modified from of
PCR which avoids a non-specific
amplification of DNA by inactivating the
taq polymerase at lower temperature.
Colony PCR
Colony PCR is used for the screening
recombinants from bacterial,
bacteriophage or yeast transformation
products.
STEPS
•Selected colonies of bacteria or yeast
are picked with a sterile toothpick or
pipette tip from a growth plate.
STEPS
• Swirl it into 25ml of TE buffer with
autoclaved H2O in a micro centrifuge tube
• Heat the mixture in water bath at 90-100c for
2 mins
• Again centrifuge it at 6000 rpm
• Collect the supernatant. Take 1-2 ml of it and
it is used as template in a 25ml PCR tube
• Conduct standard PCR
Nested PCR
Increases the specificity of DNA amplification.
Specificity of reaction enhanced by preventing
non specific binding of primers.
Nested PCR is often more successful in
specifically amplifying long DNA fragments
It requires more detailed knowledge of the
target sequences.
Nested PCR
Two pairs instead of one pair of PCR primers
are used to amplify a fragment.
First pair amplifies a fragment similar to
standard PCR.
Second pairs bind inside the 1st PCR product
fragment allow amplification of 2nd PCR
product which is shorter than the 1st one.
Advantage – very low probability of non-
specific amplification.
It is most commonly used to specifically
amplifying long DNA fragments than
conventional PCR, but it requires more
detailed knowledge of target sequences.
Touchdown PCR
This type of PCR is used to optimize yield of
amplified product at different annealing
temperature.
It is very difficult to find out the annealing
temperature when there is mismatches
between primers and the template strands.
In Touchdown PCR, gradually decreasin the
temperature till optimum temperature to be
found to anneal.
The annealing temperature is then reduced by
1C for every 1 or 2 cycles.
It is the quickest method to optimize PCR
when it is required to use new template and
primer combinations.
Nowadays, modern PCR machine which have
the facility of gradient setting are easily
programmed to run Touchdown PCR.
Multiplex PCR
Multiplex PCR is a widespread molecular
biology technique for amplification of multiple
targets in a single PCR experiment.
In a multiplexing assay, more than one target
sequence can be amplified by using multiple
primer pairs in a reaction mixture.
Generally up to eight primer pairs used in a
standard multiplex reaction, otherwise the yield
of some amplicons is reduced and not visible on
agarose gel.
RT-PCR(Reverse Transcriptase PCR)
It is a method used to amplify, isolate or
identify a known sequence from a cellular or
tissue RNA. The PCR is preceded by a
reaction using reverse transcriptase to convert
RNA to cDNA.
RT PCR procedure constitutes two
steps
First Strand Reaction-
RNA strand i.e., mRNA strand is first reverse
transcribed into ss cDNA template using dNTPs
and RNA-dependent DNA polymerase (reverse
transcriptase) through the process of reverse.
transcription.
RT PCR procedure constitutes
two steps
Second Strand Reaction-
After the cDNA is generated, standard PCR is
initiated. After ~35 cycles, millions of copies
of the sequence of interest are generated.
The original RNA template is degraded by
Rnase H leaving pure cDNA.
REAL –TIME PCR
Quantitative PCR(Q-PCR)
Itmeasure the quantity of a PCR product
(preferably real-time).
It is the method to measure starting
amounts of DNA, cDNA or RNA.
Q-PCR is used to determine whether a
DNA sequence is present in a sample and
the number of its copies in the sample.
The method with currently the highest level
of accuracy.
Cont...
Real Time PCR is based on the detection of the
fluorescence produced by a reporter molecule,
which increases, as the reaction proceeds.
This occurs due to the accumulation of the
PCR product with each cycle of amplification.
These fluorescent reporter molecules include
dyes that bind the double-stranded DNA or
sequence specific probes.
Quantitative Real-Time PCR
(qRT-PCR)
• Method use fluorescent dyes, such as Sybr
Green, probes, or fluorescence-containing
DNA such as TaqMan, to measure the
amount of amplified product as the
amplification progresses.
Progress of DNA amplification during real
time (RT-PCR) by measuring the
release of fluorescent "flashes" during
amplification.
A computer measures the rate of "flashing" in
96 simultaneous experimental PCR reactions
relative to a control reaction
Thank You
ANY QUESTION???
