introduction to PCR and its types

1. ABE Workshop 2005 July 15, 2006
Nucleic Acid Amplification Techniques

2. They are laboratory techniques that involve the
in-vitro synthesis of many copies of DNA or
RNA from one original template.

3. They are classified to
1-Target amplification
 Polymerase chain reaction(PCR)
2-Probe amplification
 Ligase chain reaction
3-Signal amplification
Branched chain signal amplification(bDNA)

5. Polymerase chain reaction
• Kary Mullis, the inventor
of PCR, was awarded
the 1993 Nobel Prize in
Chemistry

6. What is PCR?
PCR is an exponentially progressing
synthesis of the defined target DNA
sequences in vitro.

7. It is a first step in preparing DNA for :
Sequencing
RFLP
Bacterial cloning

8. What is PCR? :
Why “Polymerase”?
It is called “polymerase” because the
enzyme used in this reaction is DNA
polymerase.

9. Taq DNA polymerase
• Taq-polymerase is derived from Thermus aquaticus
(1988).
• It is Heat stable DNA polymerase

10. What is PCR? :
The “Reaction” Components
1) Target DNA - contains the sequence to be amplified.
2) Pair of Primers - oligonucleotides that
define the target. It must be Specific, their
length 18-30 nucleotides and GC content 40-
60%
3) dNTPs -
deoxynucleotidetriphosphates: DNA
building blocks.

11. 6) Buffer solution – maintains pH and ionic strength of
the reaction solution suitable for the activity of the
enzyme so concentrations rather than optimal will inhibit
Taq
5) Mg++ ions - cofactor of the enzyme
Excess magnesium gives non-specific binding
Too little magnesium gives reduced yield
4) Thermostable DNA Polymerase :-
Cycle number ranged 25-40 cycles as the half-life of Taq is
30 minutes at 95oC. Therefore if you use more than 30
cycles at denaturation times of 1 minute, the Taq will not be
very efficient at this point

12. The Reaction
THERMOCYCLERPCR tube

13. Thermal Cycling
• Typical PCR go through three steps
–Denaturation
–Annealing
–Extension

14. Denaturation
• Heating separates the double
stranded DNA at 95°c
Heat Cool

15. Annealing
• Two primers are supplied in molar excess
• They bind to the complementary region
• As the DNA cools, they wedge between two
template strands
• Optimal temperature varies based on primer length
– Typical temperature from 40 to 60 °C
– Decrease in annealing temperature result in non-
specific binding
– Increase in annealing temperature result in
reduced yield

16. Extension
 Optimal temperature 72°C
 DNA polymerase duplicates
DNA by incorporating the
four
nucleotides(Adenine,Guanine,
Thymine and Cytosine) to the
growing chain
 dNTPs follow the standard
base pairing rule

17. • The newly generated DNA strands
serve as template DNA for the next
cycle and so on.
• That’s why it is called chain

19. Types of PCR

20. PCR
RNA DNA

21. Reverse Transcriptase PCR
• Based on the process of reverse transcription, which reverse
transcribes RNA into DNA .
• First step of RT-PCR - Synthesis of cDNA from RNA template
• “Second step“-Digestion of RNA by (RNase)
• Then Standard PCR with DNA primers is performed.
RNA amplification

22. Priming RT
Oligo dT
Random hexamers
Gene specific Primers
Reverse Transcriptase
cDNA synthesis
Moloney murineleukemia
virus M-MuLV-RT (42 C)
Avian Myeloblastosis
virus AMV-RT (45 C)
Primers
Total RNA,mRNA,RNA
transcripts
Template
37-48 C

23. Removal of RNA
RNase H
RNase L
Oligonucleotide primer
Taq DNA Polymerase
PCR with gene specific
primer and Taq DNA
polymerase

24. DNA amplification
Conventional PCR
Real time PCR
Miniprimer PCR
One step
Nested PCR
Assembly PCR
Methylation mediated PCR
Ligation mediated PCR
Two steps
Multiplex PCRmultiplex
Allele specific PCR
Digital PCR
Helicase dependent amplification
Insitu PCR
Inverse PCR
Long PCR
Solid phase PCR
Touch down PCR
Hot start PCR
Modified PCR techniques

25. Real-Time PCR
Real-time PCR monitors the fluorescence emitted during
the reaction as an indicator of amplicon production at
each PCR cycle (in real time) as opposed to the endpoint
detection

26. • Miniprimer PCR: uses a polymerase that can
extend from short primers as short as 9 or 10
nucleotides. This method permits PCR
targeting to smaller primer binding regions.

27. DNA amplification
Conventional PCR
Real time PCR
Miniprimer PCR
One step
Nested PCR
Assembly PCR
Methylation mediated PCR
Ligation mediated PCR
Two steps
Multiplex PCRmultiplex
Allele specific PCR
Digital PCR
Helicase dependent amplification
Insitu PCR
Inverse PCR
Long PCR
Solid phase PCR
Touch down PCR
Hot start PCR
Modified PCR techniques

28. Nested PCR
• Two pairs of primers are used to amplify a fragment.
• First pair -amplify a fragment similar to a standard PCR.
Second pair of primers bind inside the first PCR product
fragment to allow amplification of a second PCR product
which is shorter than the first one.

30. • Assembly PCR
It is an artificial synthesis
of long DNA sequences. It
is done by performing
PCR on a pool of long
oligonucleotides with
short overlapping
segments.

31. • Methylation-specific PCR (MSP) is used to
identify patterns of DNA methylation at
cytosine-guanine (CpG) islands in genomic
DNA. Target DNA is first treated with sodium
bisulfite, which converts unmethylated
cytosine bases to uracil, which is
complementary to adenosine in PCR primers.
Two amplifications are then carried out on
the bisulfite-treated DNA: One primer set
anneals to DNA with cytosines
(corresponding to methylated cytosine), and
the other set anneals to DNA with uracil
(corresponding to unmethylated cytosine).

32. • Ligation-mediated PCR: uses small DNA
oligonucleotide 'linkers’ that are first ligated to
fragments of the target DNA. PCR primers that
anneal to the linker sequences are then used
to amplify the target fragments.

33. DNA amplification
Conventional PCR
Real time PCR
Miniprimer PCR
One step
Nested PCR
Assembly PCR
Methylation mediated PCR
Ligation mediated PCR
Two steps
Multiplex PCRmultiplex
Allele specific PCR
Digital PCR
Helicase dependent amplification
Insitu PCR
Inverse PCR
Long PCR
Solid phase PCR
Touch down PCR
Hot start PCR
Modified PCR techniques

34. • Multiplex-PCR: consists of multiple primer
sets within a single PCR mixture to
produce amplicons of varying sizes that
are specific to different DNA sequences.
Annealing temperatures for each of the
primer sets must be optimized to work
correctly within a single reaction, and
amplicon sizes should be different enough
to form distinct bands when visualized by
gel electrophoresis.

35. DNA amplification
Conventional PCR
Real time PCR
Miniprimer PCR
One step
Nested PCR
Assembly PCR
Methylation mediated PCR
Ligation mediated PCR
Two steps
Multiplex PCRmultiplex
Allele specific PCR
Digital PCR
Helicase dependent amplification
Insitu PCR
Inverse PCR
Long PCR
Solid phase PCR
Touch down PCR
Hot start PCR
Modified PCR techniques

36. Allele-specific PCR: At which primers whose 3'
ends encompass a SNP are used. so successful
amplification indicates the presence of the
specific polymorphism in the sequence.

37. • Digital polymerase chain
reaction simultaneously
amplifies thousands of
samples, each in a
separate droplet within
an emulsion.

38. • Helicase-dependent
amplification: similar to
traditional PCR, but
uses a constant
temperature rather
than cycling. DNA
helicase, an enzyme
that unwinds DNA, is
used in place of thermal
denaturation.

39. • In Situ PCR (ISH)
is a polymerase chain
reaction that actually
takes place inside the
cell on a slide or fixed
tissue .

40. Inverse PCR
• Inverse PCR is a method for
the rapid in vitro amplification
of DNA sequences that flank a
region of known sequence.
The method uses the
polymerase chain reaction
(PCR), but it has the primers
oriented in the reverse
direction of the usual
orientation. The template for
the reverse primers is a
restriction fragment that has
been ligated upon itself to
form a circle.

41. Long PCR
• Extended or longer than
standard PCR, meaning over 5
kilobases.

42. • Solid Phase PCR: where surface-bound
instead of freely-diffusing primers are used to
amplify DNA

43. Touchdown PCR (Step-down PCR): a variant of PCR
that aims to reduce nonspecific background by
gradually lowering the annealing temperature as PCR
cycling progresses. The annealing temperature at the
initial cycles is usually a few degrees (3-5°C) above the
Tm of the primers used, while at the later cycles, it is
a few degrees (3-5°C) below the primer Tm. The
higher temperatures give greater specificity for
primer binding, and the lower temperatures permit
more efficient amplification from the specific
products formed during the initial cycles.

44. Hot Start PCR
• This is a technique that
reduces non-specific
amplification during the
initial set up stages of the
PCR by inactivating the taq
polymerase at lower
temperature. i.e. specific
antibodies are used to block
Taq-polymerase at
annealing temperature.
when the temperature
raises for amplification to
72 degrees, the specific
antibody detaches from
Taq-polymerase and the
amplification with greater
specificity starts.

45. Applications of PCR
• Classification of
organisms
• Genotyping
• Mutagenesis
• Mutation detection
• Sequencing
• Cancer research
• Detection of
pathogens
• DNA fingerprinting
(ISS&VNTR)
• Drug discovery
• Genetic matching
• Genetic engineering
• Pre-natal diagnosis

46. • Intersequence-specific PCR (ISS): a method for DNA
fingerprinting that amplifies regions between
simple sequence repeats to produce a unique
fingerprint of amplified fragment lengths.

47. • Variable Number of Tandem Repeats (VNTR) PCR
targets areas of the genome that exhibit
length variation. The analysis of the genotypes
of the sample usually involves sizing of the
amplification products by gel electrophoresis.
Analysis of smaller VNTR segments known as
Short Tandem Repeats (or STRs) is the basis
for DNA Fingerprinting databases.

48. Conclusion
The speed and ease of use, sensitivity,
specificity of PCR has revolutionised
molecular biology and made PCR the
most widely used and powerful
technique with great spectrum of
research and diagnostic applications.

50. Thank you