This document describes 10 different types of PCR: 1) Conventional DNA based PCR, 2) Reverse transcription-PCR, 3) Asymmetric PCR, 4) Inverse PCR, 5) Nested PCR, 6) Anchored PCR, 7) PCR using other polymerases, 8) In situ PCR, 9) Real-Time PCR, and 10) Multiplex PCR. It provides details on the methodology and applications of each type of PCR.

1. TYPES OF PCR
1

2. Types of PCR
1. Conventional DNA based PCR
2. Reverse transcription-PCR
3. Asymmetric PCR
4. Inverse PCR
5. Nested PCR
6. Anchored PCR
7. PCR systems using other DNA polymerases
8. In situ PCR amplification and detection
9. Real-Time PCR
10.Multiplex PCR
2

3. 1. Conventional DNA based PCR
• These are the classical and conventional PCR
assays.
• The primers target sequences on DNA and
amplification follows the usual steps of
denaturation, annealing and elongation.
• Most of the PCR techniques developed for
various organisms belong to this category
3

4. 2. Reverse transcription-PCR
• m-RNA, r-RNA can be the starting material in
such types of PCR.
• It is the method used to amplify, isolate or
identify a known sequence from a cell or tissue
RNA library.
• Essentially normal PCR is preceded by reverse
transcription (to convert the RNA to cDNA).
• USES
• This is widely used in expression mapping,
determining when and where certain genes are
expressed. 4

5. 2. Reverse transcription-PCR
• Instead of Taq polymerase, Tth polymerase
from Thermus thermophilus may be used.
• Tth enzyme has both DNA polymerase and
reverse transcriptase activities at high
temperature.
• This allows both cDNA synthesis from mRNA
followed by PCR amplification.
5

6. 2. Reverse transcription-PCR
• In ordinary PCR, DNA is detected; that DNA
could be from a living or nonliving organism.
• But in reverse PCR, mRNA is detected; that
means, it is derived from a living organism.
• Presence of HIV RNA in blood can be detected
as early as 4 weeks after infection.
6

7. Viral RNA Bacterial mRNA
AAAA
3’
Protozoan (eukaryotic) poly A mRNA
Primer
Reverse transcriptase
RNA
3’
5’
5’
Extension
c
D
N
A
R
N
A
3
’
3
’
5
’
5
’
Normal PCR with two primers
7

8. 8

9. 9

10. 3. Asymmetric PCR
• This strategy has been employed for
generation of single strands for sequencing
experiments.
• By adjusting primer concentrations to favor
one strand, after 10 to 15 cycles the second
primer is used up and only strand
complementary to first strand continues to be
copied up.
10

11. 4. Inverse PCR
• Inverse PCR amplifies the stretches on either
side of a known sequence.
• Circularising a piece of DNA and hybridising
primers to the ends of known sequence in an
orientation opposite to the ends of known
sequence in an orientation opposite to
customary one, results in amplification of
primer flanking regions.
11

12. 12

13. 5. Nested PCR
• In this PCR assay, by using, a set of primers (such
as genus specific), a fragment is amplified.
• Using another set may be changing one more
primer, specific to some internal sequence
second amplification follows.
• Sensitivity and specificity is considerable
improved in nested PCR.
• PCR techniques employing this strategy have
been developed for detection of many organisms.
13

14. Semi-nested PCR
• Three primers are required, the normal
upstream and downstream primers as well as
a third, internal primer.
• Two rounds of PCR are performed, a normal
PCR with the upstream and downstream
primer, and then a second round of PCR with
the downstream and internal primer.
• A second smaller product is the result of the
second round of PCR.
14

15. Internal primer
Downstream primer
Upstream primer
Semi-nested PCR
15

16. 16

17. 6. Anchored PCR
• RACE, or Rapid Amplification of cDNA Ends, is
a technique used in molecular biology to
amplify the ends of messenger RNA (mRNA)
transcripts using a specialized reverse
transcription-polymerase chain reaction (RT-
PCR).
• It allows the amplification of an unknown end
portion of a transcript using known
information from the center of the transcript.
17

18. 6. Anchored PCR
• Uses:-
• It can be used to obtain the 5' end (5' RACE-
PCR) or 3' end (3‘ RACE-PCR) of mRNA.
• This technique is sometimes called one-sided
PCR or anchored PCR.
18

19. 19

20. 7. PCR systems using other DNA
polymerases
• (i) T7 RNA polymerase in transcription based
systems,
• (ii) DNA ligase in ligation amplification
reactions and
• (iii) Qβ replicase from bacteriophage
20

21. Ligase Chain Reaction
Probe amplification
• Probes bind immediately adjacent to one
another on template.
• The bound probes are ligated and become
templates for the binding of more probes.
• Can used to detect C. trachomatis, N.
gonorrhoeae, sickle cell mutation
21

22. Ligase Chain Reaction
Template Probes
...GTACTCTAGCT...
A G
T C
...CATGAGATCGA...
ligase
Target sequences are detected by coupled and . 22

23. Primer 1
Target
Primer 2
Target
Annealing
Ligation
Additional cycles of
denaturation, annealing, ligation
Ligase Chain Reaction
Amplification of Genomic DNA
23

24. Annealing
Ligation
Mutant Sequence
Wild-Type Sequence
DNA Product
No DNA Products
Ligase Chain Reaction Mutation Detection:
Utilizing Mutant-Specific Oligonucleotide Primers
24

25. 8. In situ PCR amplification and
detection
• Techniques have been developed for the correlation of
molecular results with cytological or histological
features.
• In these techniques, the amplification can be done
directly on the sections.
• Paraffin embedded or cytospin coated on coated glass
slides are digested with protease and amplification
solution is added.
• Taq polymerase is then added at 60 °C cycles are done.
• Product could be detected by in situ hybridisation or by
direct incorporation of biotin/digoxigenin labeled
nucleotides.
25

26. 9. REAL-TIME PCR
• The real-time PCR assay has many advantages
over conventional RT-PCR methods, including
rapidity, quantitative measurement, lower
contamination rate, higher sensitivity, higher
specificity, and easy standardisation.
• Thus, nucleic acid-based assays or real-time
quantitative assay might eventually replace virus
isolation and conventional RT-PCR as the new
gold standard for the rapid diagnosis of virus
infection in the acute-phase samples.
26

27. REAL-TIME PCR
• Real-time PCR has enhanced wider acceptance of
the PCR due to its improved rapidity, sensitivity,
reproducibility and the reduced risk of carryover
contamination.
• Real-time PCR assays used for quantitative RT-
PCR combine the best attributes of both relative
and competitive (end-point) RT-PCR in that they
are accurate, precise, capable of high throughput,
and relatively easy to perform.
27

28. REAL-TIME PCR
• Real-time PCR automates the laborious
process of amplification by quantitating
reaction products for each sample in every
cycle.
• Data analysis, including standard curve
generation and copy number calculation, is
performed automatically.
28

29. REAL-TIME PCR
• As more labs and core facilities acquire the
instrumentation required for real-time
analysis, this technique may become the
dominant RT-PCR-based quantitation
technique.
29

30. Advantages of Real-time PCR
• Rapidity due to reduced cycle times and
removal of post PCR detection procedures
• Very sensitive
• Reproducible
• Reduced risk of carry-over contamination
(sealed reactions)
• High sample throughput (~200 samples/day)
30

31. Advantages of Real-time PCR
• Easy to perform
• The detection of amplicon could be visualised
as the amplification progressed
• Allows for quantitation of results
• Software driven operation.
31

32. Real-time Reporters
1. SYBR Green,
2. TaqMan, and
3. Molecular Beacons
All real-time PCR systems rely upon the
detection and quantitation of a fluorescent
reporter, the signal of which increases in
direct proportion to the amount of PCR
product in a reaction.
32

33. 1. SYBR Green
• In the simplest and most economical format,
that reporter is the double-strand DNA-
specific dye SYBR Green (Molecular Probes).
• SYBR Green binds double-stranded DNA, and
upon excitation emits light.
• Thus, as a PCR product accumulates,
fluorescence increases.
33

34. DENATURATION STEP: DNA + PRIMERS + DYE
WEAK BACKGROUND FLUORESCENCE
ANEALING STEP:DYE BINDS dsDNA, EMITS
LIGHT
EXTENSION STEP: MEASURE LIGHT
EMMISSION 34

35. energy
fluorophore fluorophore
+ quencher
in proximity
FRET donor
FRET
acceptor
35

36. 1. SYBR Green
• The advantages of SYBR Green:-
• inexpensive,
• easy to use, and
• sensitive
36

37. 1. SYBR Green
• The disadvantage is that SYBR Green:-
• It will bind to any double-stranded DNA in the
reaction, including primer-dimers and other
nonspecific reaction products, which results in an
overestimation of the target concentration.
• For single PCR product reactions with well-
designed primers, SYBR Green can work
extremely well, with spurious nonspecific
background only showing up in very late cycles.
37

38. • The two most popular alternatives to SYBR
Green are TaqMan and molecular beacons,
both of which are hybridisation probes relying
on (FRET) forfluorescence resonance energy
transfer quantitation.
38

39. 2. TaqMan Probes
• These are oligonucleotides that contain a
fluorescent dye, typically on the 5' base, and a
quenching dye, typically located on the 3'
base.
• When irradiated, the excited fluorescent dye
transfers energy to the nearby quenching dye
molecule rather than fluorescing, resulting in a
non-fluorescent substrate.
• TaqMan probes are designed to hybridise to an
internal region of a PCR product.
39

40. 2. TaqMan Probes
• During PCR, when the polymerase replicates a
template on which a TaqMan probe is bound,
the 5' exonuclease activity of the polymerase
cleaves the probe.
• This separates the fluorescent and quenching
dyes and FRET (fluorescence resonance
energy transfer) no longer occurs.
• Fluorescence increases in each cycle,
proportional to the rate of probe cleavage.
40

41. Principle of TaqMan probe system 41

42. 3. Molecular beacons
• Also contain fluorescent and quenching dyes,
but FRET only occurs when the quenching dye
is directly adjacent to the fluorescent dye.
• Molecular beacons are designed to adopt a
hairpin structure while free in solution,
bringing the fluorescent dye and quencher in
close proximity.
42

43. 3. Molecular beacons
• When a molecular beacon hybridises to a
target, the fluorescent dye and quencher are
separated, FRET does not occur, and the
fluorescent dye emits light upon irradiation.
• Unlike TaqMan probes, molecular beacons are
designed to remain intact during the
amplification reaction, and must rebind to
target in every cycle for signal measurement.
43

44. Principle of Beacon probe system
44

45. 10. Multiplex PCR
• Use of multiple sets of primers to detect more
than one organism or to detect multiple genes
in one organism. Remember, the PCR reaction
is inherently biased depending on the G+C
content of the target and primer DNA. So
performing multiplex PCR can be tricky.
45

46. E. Coli
genome
Salmonella sp.
genome
or
10. Multiplex PCR
46

47. 10. Real-time Reporters for Multiplex
PCR
• TaqMan probes and molecular beacons allow
multiple DNA species to be measured in the
same sample (multiplex PCR), since
fluorescent dyes with different emission
spectra may be attached to the different
probes.
47

48. 10. Real-time Reporters for Multiplex
PCR
• Multiplex PCR allows internal controls to be
co-amplified and permits allele discrimination
in single-tube, homogeneous assays.
• These hybridisation probes afford a level of
discrimination impossible to obtain with SYBR
Green, since they will only hybridise to true
targets in a PCR and not to primer-dimers or
other spurious products.
48

49. Viral Quantitation
• The majority of diagnostic PCR assays
reported to date have been used in a
qualitative, or ‘yes/no’ format.
• The development of real-time PCR has
brought true quantitation of target nucleic
acids out of the pure research laboratory and
into the diagnostic laboratory.
49

50. Viral Quantitation
• Determining the amount of template by PCR
can be performed in two ways:
1. Relative quantitation and
2. Absolute quantitation
1. Relative quantitation: Describes changes in
the amount of a target sequence compared
with its level in a related matrix.
50

51. Viral Quantitation
1. Relative quantitation:-
• Generally, relative quantitation provides
sufficient information and is simpler to
develop.
• However, when monitoring the progress of an
infection, absolute quantitation is useful in
order to express the results in units that are
common to both scientists and clinicians and
across different platforms.
51

52. Viral Quantitation
• Absolute quantitation may also be necessary
when there is a lack of sequential specimens
to demonstrate changes in virus levels, no
suitably standardised reference reagent is
available or when the viral load is used to
differentiate active versus persistent infection.
52

53. Viral Quantitation
• 2. Absolute quantitation: States the exact
number of nucleic acid targets present in the
sample in relation to a specific unit.
• A very accurate approach to absolute
quantitation by PCR is the use of competitive co-
amplification of an internal control nucleic acid
of known concentration and a wild-type target
nucleic acid of unknown concentration, with the
former designed or chosen to amplify with an
equal efficiency to the latter.
53

54. • However, while conventional competitive PCR is
relatively inexpensive, real-time PCR is far more
convenient, reliable and better suited to quick
decision making in a clinical situation.
• This is because conventional, quantitative,
competitive PCR (qcPCR) requires significant
development and optimisation to ensure
reproducible performance and a predetermined
dynamic range for both the amplification and
detection components.
54

55. Limitations of Real-time PCR
• Inability to monitor amplicon size without
opening the system.
• Incompatibility of some platforms with some
fluorogenic chemistries.
• The relatively restricted multiplex capabilities
of current applications.
• The start-up expense of real time PCR is
prohibitive when used in low-throughput
laboratory.
55

56. CONCLUSIONS
• Advances in the development of fluorophores,
nucleotide labeling chemistries, and the novel
applications of oligoprobe hybridisation have
provided real-time PCR technology with a
broad enough base to ensure its acceptance.
• Recently, instrumentation has come up that is
capable of incredibly short cycling times
combined with the ability to detect and
differentiate multiple amplicons.
56

57. CONCLUSIONS
• New instruments are also flexible enough to
allow the use of any of the chemistries making
real-time nucleic acid amplification an
increasingly attractive and viable proposition for
the routine diagnostic laboratory.
• In many cases these laboratories perform tissue
culture to isolate virus and serological methods to
confirm the identity of the isolate, which may
take a considerable, and clinically relevant,
amount of time.
57

58. References
• Textbook of Medical Biochemistry By Dr. MN
Chatterjee and Dr. Rana Shinde
• Textbook of Medical Biochemistry, Sixth
Edition, By DM Vasudevan
58

59. References
• http://www.worldofteaching.com
• http://biotools.umassmed.edu/bioapps/primer3
_www.cgi
• http://arbl.cvmbs.colostate.edu/molkit/rtranslat
e/index.html
•http://www.genscript.com/custom_service.html?
&gs_cust=391826&gs_camp=316
•http://www.bio.davidson.edu/courses/genomics/
chip/chip.html
59

60. References
• http://www.genscript.com/custom_service.ht
ml?&gs_cust=391826&gs_camp=316
• http://www.bio.davidson.edu/courses/genomi
cs/chip/chip.html
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61. THANK YOU
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