for MBBS Students by Dr vishnu Kumar

1. Dr. Vishnu Kumar
Professor, Department of
Biochemistry, SRMSIMS, Bareilly
vkawasthi@hotmail.com
madhwapur1976@gmail.com
PCR & RT - PCR

2. 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

3. 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

4. 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.

5. Requirements.
• 1) Target DNA template.
• 2) Oligo nucleotide primers.
• 3) Taq Polymerase.
• 4) dNTPs.
• 5) Reaction Buffer.
• 6) Mineral oil.

7. 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.

10. Denaturation
• The isolated duplex DNA containing the
target sequence is heated to 95° C for 1
minute to separate the two strands.

11. Annealing of primers.
• The mixture is cooled to 45°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.

12. 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.

13. 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.

14. 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.

15. 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.

16. 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.

17. RealTime-PCR

18. 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.

19. 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)

20. Real Time PCR Instruments

21.  LightCycler (Idaho Technologies Roche)
 Rotor-Gene (Corbett Research)
 iCycler (BioRad)
 Mx4000™ Multiplex Quantitative PCR System
 ABI Prism 7700 (Perkin-Elmer-Applied-Biosystem)
 SmartCycler (Cephid)
Instruments

22. 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

23. Real Time Detection
1a. Excitation filters 1b.
Emission filters
Tungsten halogen light source
(350 - 1000nm continuous)
Microplate format
Cycler
iCycler from BioRad

24. Probe types & Design

25. dsDNA BindingDye
 SYBR Green I
 SYBR Green II
 EVAGreen
 LC Green
 BEBO
 YO-PRO
 SYTO family

26. 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

27. Hydrolysis Probes (TaqMan)

28. 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

29. 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 Tth cleaves off the reporter
Reporter is separated from the quencher.
Fluorescent signal
Signalis proportional to the
amount of amplified product in the
sample
TaqMan Probe

30. 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
TaqMan Probe

31. Loop
Stem
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.
Hairpin probes: Molecular beacons

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

34. 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)

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

36. The primer is
part of the
Scorpion probe
The primer is
extended
The template &
probe denature
The primer binds
to the target
Scorpion stem-loop
format
Primer, stopper
toread PCR through,
prevent
probe
sequence, fluorophore &
quencher (detection system).
The probe binds to the
complimentary sequence
of the DNA

37. 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

38. Hybridization probes
Probe 2
Probes hybridize to their
target sequences in a
head-to-tail arrangement. FRET
Probe 1

39. Hybridization probes
h
D
A
h
A
FRET
D
Amplicon
Probe 1
Probe 2
D FAM A LC red 640

40. 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 AD
FRET

41. 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
Application in Molecular
Diagnostics

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

43. SHORT ANSWER QUESTIONS
1.Taq Polymerase
2. Denaturation of DNA
3.Applications of RT - PCR
4.Advantage and Disadvantage of P.C.R.

44. 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

45. 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