UIMLT

 Prepared by :Syeda Rida Shah

 Senior lecturer
 THE UNIVERSITY OF LAHORE
Types of Polymerase Chain Reaction
PCR
Types
 Allele-specific PCR
 Hot-start PCR
 Tochdown PCR
 Realtime PCR
 RT –PCR
 Multiplex PCR
 Nested PCR
 Types

 Inverse PCR
 In situ PCR
 Long PCR
 Colony PCR
 Allele-specific PCR
 This diagnostic or cloning technique is used
to identify or utilize single-nucleotide
polymorphisms(SNPs) (single base
differences in DNA).
 It requires prior knowledge of a DNA
sequence, including differences between
alleles
 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.
0 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 then be extended by the Taq DNA
polymerase, generating nonspecific products and
lowering product yields.

0 The hot start PCR is a modified form of

Polymerase chain reaction (PCR) which avoids a
non-specific amplification of DNA by
inactivating the taq polymerase at lower
 Colony PCR

0Colony 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.
• Swirl it into 25micro l of TE buffer with
autoclaved d H2O in an micro centrifuge tube
• Heat the mix in boiling water bath at 90-100c for 2
mins
• Again centrifuge it at 6000 rpm
• Collect the supernatant. Take 1-2 micro l of it and
it is used as template in a 25micro l PCR tube
• Conduct standard PCR
 Nested PCR
 Increases the specificity of DNA
amplification.
 Nested PCR is often more successful in
specifically amplifying long DNA fragments
 It requires more detailed knowledge of the
target sequences.
 Nested PCR

0 Two pairs instead of one pair of PCR

primers are used to amplify a fragment.
0 First pair amplifies a fragment similar to
standard PCR.
0 Second pairs bind inside the 1st PCR
product fragment allow amplification of
2nd PCR product which is shorter than the
1st one.
0 Advantage – very low probability of
non-specific amplification.
0 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, the Ta during the first 2
cycles is set at ~3C above the calculated Ta. The
annealing temperature is then reduced by 1C for
every 1 or 2 cycles.
Cont.…

0 It is the quickest method to optimize PCR

when it is required to use new template and
primer combinations.

0 Nowadays, modern PCR machine which have

the facility of gradient setting are easily
programmed to run Touchdown PCR.
 Multiplex PCR

 0 Multiplex PCR is a widespread molecular biology

technique for amplification of multiple targets in a
single PCR experiment.
 0 In a multiplexing assay, more than one target

sequence can be amplified by using multiple primer

pairs in a reaction mixture.
 0 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.
MULTIPLEX
PCR
 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
0 First Strand Reaction-
RNA strand i.e., mRNA strand is
first reverse transcribed into ss
RNA- cDNA dependent
template DNA using
polymerase
dNTPs and (reverse
process of
transcriptase) through the reverse
transcription.
0 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.
 Quantitative PCR(Q-PCR)

 It measure 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.
 Real –time PCR
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, amount 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
Uses for PCR

 Research
 Gene cloning
 Clinical
 DNA fingerprinting

 Crime scene analysis

 Real-time PCR
 Paternity testing

 DNA sequencing
 Genetically inherited
diseases
 Applications
Genome study
• Quantitation of gene expression
• DNA damage (microsatellite instability) measurement
• Detection of inactivation of gene at X-chromosome
• Genotyping
Pathogen detection
• viral quantitation
• Bacterial
 Cont…
monitoring
• Drug therapy efficacy / drug monitoring
• Prenatal diagnosis
• cancer diagnostics
 Cont…
PCR is being used for detection, genotyping and
quantification of the disease-causing agents like
•hepatitis C virus,
•hepatitis B virus
•cytomegalovirus
•Mycobacterium tuberculosis.
•prenatal diagnosis of disorders like β-thalassaemia, α-

thalassaemia,
•sickle cell disorders, trisomy 21, trisomy 18,
•Haemophilia
•cystic fibrosis,
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