PCR & RT – PCR

Competency Number BI: 7.4

CORE COMPETENCY

Dr. Vishnu Kumar

Professor, Department of
Biochemistry, HIMS, Bareilly
vkawasthi@hotmail.com
madhwapur1976@gmail.com
 Learning Objectives
After completion of this lecture learner
should be able to define :

 PCR & RT PCR

 Components of PCR & RT - PCR
 Steps of PCR & RT PCR
 Applications of PCR & RT PCR
Advantage & Disadvantage of PCR & RT
PCR
 Types of the PCR
• Traditional or Conventional PCR Or PCR
• Real time PCR

• Colony PCR
• Nested PCR
• Multiplex PCR
• AFLP PCR
• Hot Start PCR
• In Situ PCR
• Inverse PCR
• Asymmetric PCR
• Long PCR
• Long Accurate PCR
• Reverse Transcriptase PCR
• Allele specific PCR
 Colony PCR
Colony PCR- the screening of bacterial (E.Coli) or yeast clones for correct ligation
or plasmid products.

Pick a bacterial colony with an autoclaved toothpick, swirl it into 25 μl of TE

autoclaved dH2O in an microfuge tube.

Heat the mix in a boiling water bath (90-100C) for 2 minutes Spin sample for 2

minutes high speed in centrifuge.

Transfer 20 μl of the supernatant into a new microfuge tube

Take 1-2 μl of the supernatant as template in a 25 μl PCR standard PCR

reaction.
 Hot Start PCR
• This is a technique that reduces non-specific
amplification during
the initial set up stages of the PCR

• The technique may be performed manually by

heating the reaction components to the
melting temperature (e.g., 95°C) before adding
the polymerase
• Specialized enzyme systems have been
developed that inhibit the polymerase's
activity at ambient temperature, either by the
binding of an antibody or by the presence of
covalently bound inhibitors that only
dissociate after a high-temperature activation
step

• DNA Polymerase- Eubacterial type I DNA

polymerase. These thermophilic DNA
polymerases show a very small polymerase
activity at room temperature.
 Asymmetric PCR
• Asymmetric PCR is used to preferentially amplify one
strand of the original DNA more than the other.
• It finds use in some types of sequencing and
hybridization probing where having only one of the two
complementary stands is ideal.
• PCR is carried out as usual, but with a great excess of one
primers for the chosen strand.
 AFLP PCR
AFLP is a highly sensitive PCR-based
method for detecting polymorphisms in
DNA. AFLP can be also used for
genotyping individuals for a large
number of loci
• Genomic DNA is digested with one or more
restriction enzymes. tetracutter (MseI) and a
hexacutter (EcoRI).

• Ligation of linkers to all restriction fragments.

• Pre-selective PCR is performed using primers

which match the linkers and restriction site
specific
sequences.

• Electrophoretic separation and amplicons on a gel

matrix, followed by visualisation of the band
pattern.
 Inverse PCR
• Inverse PCR (Ochman et al., 1988) uses standard PCR
(polymerase chain reaction)- primers oriented in the reverse
direction of the usual orientation.
• The template for the 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.
Applications
• Amplification and identification of sequences
flanking transposable elements, and the identification of
genomic inserts.
 Multiplex PCR
• Multiplex PCR is a variant of PCR which enabling
simultaneous amplification of many targets of interest in
one reaction by using more than one pair of primers.
 In Situ PCR
• In Situ PCR (ISH) is a polymerase chain reaction that actually
takes place inside the cell on a slide. In situ PCR amplification
can be performed on fixed tissue or cells.

• Applies the methodology of hybridization of the nucleic acids.

• Allows identification of cellular markers

• Limited to detection of non-genomic material such as RNA,

genes or genomes
In Situ
PCR
 Long PCR
• Extended longer than standard PCR, meaning over 5
kilobases
or (frequently over 10 kb).

• Long PCR is useful only if it is accurate. Thus, special mixtures

of proficient polymerases along with accurate polymerases
such as Pfu are often mixed together.

• Application- to clone large genes not possible with

conventional PCR.
 Polymerase chain reaction
(Conventional/ Traditional PCR)
• It is a technique for multiplying a single
template of DNA. It provides a sensitive,
selective and rapid means of amplifying any
desired sequence of DNA.
• This is a test tube method of amplifying a
selected DNA sequence. Invented by Kary
Mullis in 1984.
• It permits synthesis of millions of copies of
DNA sequence within a few hours.
 Requirements.
• 1) Target DNA template.
• 2) Oligo nucleotide primers.

• 3) Taq Polymerase.
• 4) dNTPs.

• 5) Reaction Buffer.
• 6) Mineral oil.
 Steps of P. C. R.
• 1) Denaturation.
• 2) Annealing of primers.
• 3) Elongation or Extension.

• The average P. C. R. involves 30- 35

cycles of reactions that provide
sufficient copies of the original DNA.
 Denaturation
• The isolated duplex DNA containing the
target sequence is heated to 95° C for
1 minute to separate the two strands.
 Annealing of primers.
• The mixture is cooled to 55°C. cooling
is due to better annealing with the
primers.

• The primer is added in vast excess.

• A large excess of primer is added to

ensure that they promptly anneal with
the flanking sequences.
 Elongation.

• Each primer is elongated by DNA (Taq)

polymerase in presence of dATP, dGTP,
dCTP and TTP. Each of the two DNA
strands serve as a template for the
synthesis of new DNA from the two
primers. Temperature is increased to
72° C for polymerization.
 Taq Polymerase.
• Normal DNA polymerase will be
destroyed by each heat denaturation
cycle.
• So substitution of a heat stable DNA
polymerase called Taq polymerase is
used. Taq polymerase is an enzyme
present in thermus aquaticus, an
organism that lives in and replicates in
hot spring at 70° to 80° C.
 Advantages of P.C.R.
• 1) Less expensive and quicker than the
biological method.

• 2) More sensitive, even nano gram

quantity can be amplified.

• 3)Badly degraded DNA e.g. DNA from

formalin fixed tissue can be amplified.
 Disadvantages of P.C.R.
• 1) Error rate is high as Taq polymerase
does not have proof reading activity.

• 2) Small sequence can be amplified

( 200-2000) base pairs, can not reliably
amplify larger sequences.
 Application.
• 1) Prenatal diagnosis of genetic
diseases.
• 2) Detection of criminal from a drop of
blood or a small strand of hair.
• 3) Paternity test.
• 4) Bacterial, viral or protozoal diseases
can be diagnosed before they could
manifest clinically, e.g. tuberculosis at
very early stage.
RealTime-PCR
 What is Real Time PCR?

Real Time PCR is a technique in which

fluoroprobes bind to specific target regions of
amplicons to produce fluorescence during PCR.
The fluorescence, measured in Real Time, is
detected in a PCR cycler with an inbuilt filter
flurometer.
 History of Real Time PCR
Initial work by Higuchi and first demonstrated the
simultaneous amplification and detection of specific
DNA sequences in real- time by simply adding
ethidium bromide (EtBr) to the PCR reaction so that
the accumulation of PCR product could be visualised
at each cycle. (Higuchi et al., 1992)
 RT PCR Instruments

 LightCycler (Idaho Technologies Roche)

 Rotor-Gene (Corbett Research)
 iCycler (BioRad)
 Mx4000™ Multiplex Quantitative PCR System
 ABI Prism 7700 (Perkin-Elmer-Applied-Biosystem)
 SmartCycler (Cephid)
 General Description of Instruments

1. PCR cycler:
1. 96 well format, 8 tube format, capillary (glass)
2. Air or block heater
3. Temperature ramp, temperature gradient
2. Fluorescence emission & detection :
1. Fluorometer
2. CCD camera
3. Excitation source: xenon, halogen, laser

3. Fluorescent Dye Labeling of:

1. Oligonucleotides
Real Time Detection

1a. Excitation filters 1b.

Emission filters
Tungsten halogen light source
(350 - 1000nm continuous)
Microplate format
Cycler

iCycler from BioRad

Probe types & Design
 dsDNA BindingDye
 SYBR Green I

 SYBR Green II

 EVA Green

 LC Green

 BEBO

 YO-PRO

 SYTO family
Sybr Green PCR Assay
 Stronger signal
 Higher selectivity for dsDNA
 Lesser sequence dependent
 Higher stability
 Lesser inhibitory for Taq
 Higher resolution in melting curves
 Less hazardous and mutagenicity)

Binds to
 Non specific PCR product
 Primer dimer
Hydrolysis Probes (TaqMan)
What is Fluorescence Resonance Energy
Transfer (FRET)?
FRET is a distance dependent interaction
between the excited states of 2
dye molecules in which
excitation is transferred from a
donor molecule to an acceptor
molecule without emission of a
photon
TaqMan Probe
 When intact, the fluorescence of the reporter
is quenched due to its proximity to
the
quencher
 Probe hybridizes to the target

dsDNA-specific 5'—>3' exonuclease activity

of Taq or cleaves off the reporter

Reporter is separated from the quencher.

Fluorescent signal
Signal is proportional to the
amount of amplified product in the
sample
 TaqMan Probe
Advantages
 Highly fluorogenic

 Easy PCR setup

 Sequence-specific detection, multiplexing

Disadvantages
 Expensive

 Probe design and positioning challenging

 Similar conditions for primers and probes

 Elevated background (Quenching capacity)

 Probe degraded: no end-point analysis

 Hairpin probes: Molecular beacons
Molecular Beacons are hairpin structures composed of a (25–40 nt)
nucleotide base paired stem and a target specific nucleotide loop.

The loop consists of target specific nucleotide (probe) sequences (15–30 nt)

A fluorescent moiety (reporter)is attached to 5’ end and a quencher moiety is

attached to 3’end. The stem keeps both the moieties in close proximity so
that fluorescence is quenched.

Loop

Stem
5’ 3’
3’ 5’

 Operation of Molecular
Denaturation
(MB): MB is non-fluorescent due to
Beacon
5’ 3’
close proximity of the non-
5’
5’
fluorescent quencher (Q) the
R Q
and
fluorescent Reporter
3’ 5’
The probe denatures and the loop
Primer molecular anneals to the target sequence of
Beacon
5’ annealing the amplicon
3’
Separating the quencher from the
5’ fluorophore and thereby producing
3’ 5’
fluorescence which is proportional to
the amplicons produced during PCR
Extension
MB is displaced not destroyed
5’ 3’
5’
during amplification, because a DNA
polymerase lacking 5' exonuclease
activity is used
5’
3’ 5’
Q
 Molecular beacons
Advantages
 High specificity, low background

 Post PCR analysis

 PCR multiplex

 Allelic discrimination (greater specificity than linear

probes)

Disadvantages
 Challenging design

 Long probes – less yield

 Intramolecular competitive binding

 Low signal levels (proximity of reporter and quencher)

 Scorpion Primers
Complementary sequence
Scorpion primer consists of:
PCR
primer
3’ Quencher
5’ Reporter
Blocker

The loop of the Scorpions probe includes a sequence that is complementary to

an internal portion of the sequence it primes.
During the first amplification cycle, the Scorpions primer is extended, and the
sequence complementary to the loop sequence is generated.
After subsequent denaturation and annealing, the loop of the Scorpions probe
hybridizes to the internal target sequence, and the reporter is separated from the
quencher. The resulting fluorescent signal is proportional to the amount of
amplified product in the sample.
The Scorpions probe contains a PCR blocker just 3' of the quencher to prevent
read-through during the extension of the opposite strand.
 Hybridization Probes
 These assays use two sequence-specific oligonucleotide probes in
addition to two sequence specific primers. The two probes are
designed to bind to adjacent sequences in the target. The probes are
labeled with a pair of dyes that can engage in FRET. The donor dye is
attached to the 3' end of the first probe, while the acceptor dye is
attached to the 5' end of the second probe.

 During real-time PCR, excitation is performed at a wavelength specific

to the donor dye, and the reaction is monitored at the emission
wavelength of the acceptor dye. At the annealing step, the probes
hybridize to their target sequences in a head-to-tail arrangement. This
brings the donor and acceptor dyes into proximity, allowing FRET to
occur.

 The increase in PCR product is proportional to amount of fluorescence

 Hybridization probes
Advantages
– Probe with only one fluorophore

– Easy synthesis and quality controls

– Reduced background fluorescence

– High specificity

Disadvantages
• Strict compatibility between donor & acceptor
fluorophores

h D
FRET
A
 Application in Molecular
Diagnostics
 Clinical microbiology and Food microbiology
 Gene expression
 viral quantitation
 Single Nucleotide Polymorphism (SNP) analysis
 Clinical oncology
 Cancer
 Analysis of cellular immune response in peripheral blood
 Chromosome aberrations
 LONG ANSWER QUESTIONS

 Describe the PCR in detail and its

application.
 Describe the RT - PCR in detail and its
application.
 SHORT ANSWER QUESTIONS

1.Taq Polymerase
2. Denaturation of DNA
3.Applications of RT - PCR
4.Advantage and Disadvantage of P.C.R.
 MCQ PCR, RT PCR & its Applicatipon

1. The DNA PROBE IS RADIOLABELLED BY

a. In situ hybridization
b. Southern blotting
c. Nick translation
d. Restriction mapping
Answer: c
2. The technique used to produce a cDNA is
prepared by using the enzyme:

a. RNA Polymerase
b. DNA Polymerase
c. Reverse transcriptase
d.Restriction Endonuclease
Answer: c