1. by : Mahdi zarei
M.Sc. Student ,clinical biochemistry
Ferdowsi university of mashhad

2. History:
• By 1971 researchers in Khorana's project, concerned over their yields of DNA, began
looking at "repair synthesis" - an artificial system of primers and templates that allows
DNA polymerase to copy segments of the gene they are synthesizing. Although similar
to PCR in using repeated applications of DNA polymerase, the process they usually
describe employs just a single primer-template complex, and therefore would not lead
to the exponential amplification seen in PCR.
• Kary Mullis is generally credited with inventing PCR in 1983 while working for Cetus
Corporation in Emeryville, California. While driving on Highway 128 from San
Francisco to Mendocino, Mullis made an intellectual leap. He reasoned that by using
two opposed primers, one complementary to the upper strand and the other to the
lower, then performing multiple cycles of denaturation, annealing and polymerization
he could exponentially amplify the piece of DNA between the primers.

3.  PCR was invented in 1983 by ( Kary mullis ) & he received the
Nobel Prize in chemistry in 1993, for his invention.
 It revolutionized biological methods specially in molecular
cloning in a way that it has became an inseparable & irreplaceable
part of molecular investigations.
3

4. DEVELOPMENT OF PCR

5. Development….
 PCR work was first published (1985)using Klenow polymerase
unstable with heat
New enzyme had to be added manually at each step
Maximum length 400bp
– not very practical
 First reports using DNA polymerase
from Thermus aquaticus (1988)
• Taq-polymerase (Saiki et al, 1988) from
Yellowstone National Park hot springs

6. Thermostable Polymerases
Polymerase
Taq pol
Amplitaq
(Stoffel
fragment)
Vent*
T ½,
95oC
40 min
Extension Type of
Rate (nt/sec) ends
75
3’A
80 min
>50
3’A
400 min
>80
95%
blunt
Blunt
Source
T. aquaticus
T. aquaticus
Thermococcus
litoralis
Pfu
>120 min
60
Pyrococcus
furiosus
Tth*
20 min
>33
3’A
T.
(RT activity)
thermophilus
*Have proof-reading functions and can generate products over 30
kbp

7. Automation of PCR
Developed automatic “thermocycler” programmable heat block…
• The early PCR experiments , researchers had to rely on a series of water baths to
maintain the different temperatures required by the procedure . “cycling” involved
manual transfer of samples from one water bath to another at specified times.
• In 1988, perkin-elmer introduced the thermal cycler , a revolutionary device that
automatically and repetitively raised and lowered the temperature of the samples
during PCR cycles . This allowed the PCR technique to be automated. Subsequent
refinements of this device extended the flexibility and accuracy of pcr.
• While in some old machines the block is submerged in an oil bath to control
temperature, in modern PCR machines a Peltier element is commonly used.

8. PCR Thermocycler

9. Stage of pcr
 Exponential amplification:
 At every cycle, the amount of product is doubled
(assuming 100% reaction efficiency). The reaction is
very sensitive.
 Leveling off (linear)stage:
 The reaction slows as the DNA polymerase loses
activity and as consumption of reagents such as
dNTPs and primers causes them to become
limiting.
 Plateau:

No more product accumulates due to exhaustion
of reagents and enzyme.

10. COMPONENT OF PCR REACTION
&
PCR PROCEDURE

11. Components of PCR Reaction
• Template DNA
• Flanking Primers
• Thermo-stable polymerase
• Taq Polymerase
• dNTP
• (dATP, dTTP, dCTP, dGTP)
• PCR Buffer (mg++)
• Thermocyler
Thermus aquaticus

12. primers
1. PCR primers should be 10-24 nucleotides in length.
2. The GC content should be 40%-60%.
3. The primer should not be self-complementary or complementary
to any other primer in the reaction mixture, to prevent primer-dimer and
hairpin formation.
4. Melting temperatures of primer pairs should not differ by more than 5°C, so
that the GC content and length must be chosen accordingly.
5. The melting and annealing temperatures of a primer are estimated as
follows: if the primer is shorter than 20 nucleotides, the approximate melting
temperature is calculated with the formula:
Tm = 4(G + C) + 2 (A + T)
6. The annealing temperature should be about 5°C lower than the melting
temperature.

13. PCR Buffer
 Basic Components
 20mM Tris-HCL pH 8.4
 50mM KCl
 1.5 mM MgCl2
 Magnesium – Since Mg ions form complexes with dNTPs, primers and DNA templates, the
optimal concentration of MgCl2 has to be selected for each experiment. Too few Mg2+ ions result in
a low yield of PCR product, and too many increase the yield of non-specific products and promote
mis incorporation.
 Potential Additives
 Helix Destabilisers - useful when target DNA is high G/CWith NAs of high (G+C) content.
 dimethyl sulphoxide (DMSO),
 dimethyl formamide (DMF),
 urea
 formamide
 Long Targets >1kb. Formamide and glycerol
 Low concentration of template: Polyethylene glycol (PEG)

14. Temperature
 Denaturation
 Trade off between denaturing DNA and not denaturing Taq Polymerase
 Taq half-life 40min at 95 , 10min at 97.5
 95
 Annealing
 Trade off between efficient annealling and specificity
 2-5 below Tm
 Extension
 Temperature optimum for Taq Polymerase
 72

15. Temperature
100
PCR
Melting
94 oC
50
0
T i m e
3’
5’
5’
3’

16. Temperature
100
PCR
Melting
94 oC
50
0
T i m e
3’
5’
Heat
5’
3’

17. Temperature
100
Melting
94 oC
50
Melting
o
Extension 94 C
Annealing
72 oC
Primers
50 oC
0
T i m e
3’
5’
5’
5’
5’
3’
PCR

18. Temperature
100
Melting
94 oC
50
Melting
o
Extension 94 C
Annealing
72 oC
Primers
50 oC
0
T i m e
3’
5’
Heat
5’
5’
Heat
5’
5’
3’
30x
PCR

19. Temperature
100
Melting
94 oC
50
Melting
o
Extension 94 C
Annealing
72 oC
Primers
50 oC
0
T i m e
3’
5’
5’
5’
5’
5’
5’
5’
5’
3’
30x
PCR

20. Temperature
100
50
0
3’
5’
5’
Melting
94 oC
Melting
o
Extension 94 C
Annealing
72 oC
Primers
50 oC
T i m e
5’
5’
5’
3’
Heat
5’
5’
Heat
5’
30x
PCR

21. Temperature
100
50
0
3’
5’
5’
Melting
94 oC
Melting
o
Extension 94 C
Annealing
72 oC
Primers
50 oC
T i m e
5’
5’
5’
5’
3’
5’
5’
5’
5’
5’
5’
30x
PCR

22. Temperature
100
Melting
94 oC
50
0
3’
5’
5’
Melting
o
Extension 94 C
Annealing
72 oC
Primers
50 oC
T i m e
5’
5’
5’
5’
3’
5’
Fragments of
defined length
5’
5’
5’
5’
5’
30x
PCR

23. More Cycles = More DNA
Size Number of cycles
Marker 0 10 15 20 25 30

24. Detection of pcr product
Detection
Visualization
Agarose gel and/or polyacrylamide gel
electrophoresis
-EtBr staining (UV transilluminator, image
analyzer)
-Southern blotting (hybridization with labeled
probe)
-Silver staining
Restriction endonuclease digestion
-Agarose or polyacrylamide gel -HPLC
Dot blots
Hybridization with labeled probe
High-pressure liquid chromatography
UV detection
Electrochemiluminescence
Voltage-initiated chemical reaction/photon
detection
Direct sequencing
Radioactive or fluoescent-based DNA
sequencing

25. Controls for PCR
 Blank reaction
 Controls for contamination
 Contains all reagents except DNA template
 Negative control reaction
 Controls for specificity of the amplification reaction
 Contains all reagents and a DNA template lacking the target sequence
 Positive control reaction
 Controls for sensitivity
 Contains all reagents and a known target-containing DNA template

26. Interpretation of the PCR Results
 The PCR product should be of the expected size.
 No product should be present in the reagent blank.
 Misprimes may occur due to non-specific hybridization of primers.(pcr product
present in the negative control)
 Primer dimers may occur due to hybridization of primers to each other.

27. Variations of the PCR

28. Hot Start PCR
 It is a method for increasing specificity of PCR reactions.
 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
 DNA polymerase- eubacterial type I DNA polymerase, Pfu
 Ampliwax or antibody are used in hot start pcr

30. Nested PCR
 It is a method for increasing specificity of PCR reactions.
 Two pairs (instead of one pair) of PCR primers are used to amplify a fragment.
 First pair -amplify a fragment similar to a standard PCR. Second pair of primers-
nested primers - bind inside the first PCR product fragment to allow
amplification of a second PCR product which is shorter than the first one.
 Advantage:
Very low probability of nonspecific amplification

32. Touchdown PCR
 It is a method for increasing specificity of PCR reactions.
 Touchdown PCR uses a cycling program where the annealing temperature is
gradually reduced (e.g. 1-2°C /every second cycle). The initial annealing
temperature should be several degrees above the estimated Tm of the primers.
The annealing temperature is then gradually decreased until it reaches the
calculated annealing temperature of the primers or some degrees below.
Amplification is then continued using this annealing temperature.

34. 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.
 Application :
Amplification and identification of flanking sequences such as
transposable elements, and the identification of genomic inserts.

36. Reverse Transcriptase PCR
• Based on the process of reverse transcription, which reverse
transcribes RNA into DNA and was initially isolated from
retroviruses.
• First step of RT-PCR - "first strand reaction“-Synthesis of cDNA
using oligo dT primers (37°C) 1 hr.
• “Second strand reaction“-Digestion of cDNA:RNA hybrid
(RNaseH)-Standard PCR with DNA oligo primers.
• Allows the detection of even rare or low copy mRNA sequences by
amplifying its complementary DNA.

37. 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.

38. Real-time PCR
 real-time polymerase chain reaction, also called quantitative real time
polymerase chain reaction (qPCR) or kinetic polymerase chain reaction.
 Real-time PCR detects and measures the amplification target DNA as they
are produced.
 Unlike, conv. PCR, real-time PCR uses an oligonucleotide probe labeled
with fluorescent dyes or an alternative chemistry, and a thermocycler
equipped with the ability to measure fluorescence.

39. Real time PCR in comparison with other technical
methods
 amplification can be monitored real-time
 no post-PCR processing of products
(high throughput, low contamination risk)
 Less time to getting results
 No gel-based analysis at the end of the pcr reaction
 Computer based analysis of the cycle-fluorescence time course
 most specific, sensitive and reproducible
 not much more expensive than conventional PCR
(except equipment cost)

40. The Basic of Real time PCR

41. Two method for quantify real time pcr results:
ABSOLUTE QUANTIFICATION
&
RELATIVE QUANTIFICATION

42. Absolute quantification

43. Relative quantification
 This involves comparing the Ct values of the samples of interest with a control or
calibrator such as a non-treated sample or RNA from normal tissue. The Ct values
of both the calibrator and the samples of interest are normalized to an appropriate
endogenous housekeeping gene(GAPDH ,rRNA ,…).
 The comparative Ct method is also known as the 2-ΔΔCt method :
ΔΔct=Δct sample – Δct reference
Here, Δct,sample is the Ct value for any sample normalized to the endogenous
housekeeping gene and ΔCt, reference is the Ct value for the calibrator also normalized
to the endogenous housekeeping gene.

44. Detection in real time PCR
 Uses fluorescence as a reporter by Three general methods :
1. DNA-binding agents
(SYBR Green)less accuracy.
2. Hydrolysis probes(TaqMan)
3. Hybridization probes
(Light Cycler) most accurate & specific.
Beacons, Scorpions

45. ®
1.SYBR



green
DNA binding dye
Binds to minor groove (dsDNA)
Emits light when bound
More double stranded DNA = more
binding = more fluorescence
 Forensically, can be used to calculate how
much DNA was present before reaction.



Unspecific
Melting curve analysis

46. The advantage of this technique is that it is relatively cheap as it can
be used with any pair of primers for any target. However, as the
presence of any dsDNA generates fluorescence, specificity of this
assay is greatily decrease due to amplification of nonspescific PCR
products and primer-dimers. Generating and comparing melting
curves using the light cycler is one method of increasing the
specificity of the reaction.

47. Melting curve analysis

49. Hydrolysis probe(taqman)

50. Dual hybridization probe

51. Molecular beacon

52. scorpions

53. references
 MT Rahman , MS Uddin , R Sultana , A Moue , M Setu . Polymerase chean
reaction (PCR) : A Short Rewiew . AKMMC J 2013: 4(1): 30-36
 Manit A,Iqubal S ,Magali W ,Lyndon G et all .basic principles of real-time
quantification pcr .mol.diagn.5(2),209-219.(2005)
 http://www.nature.com/nprot/journal/v1
 http://www.cryst.bbk.ac.uk/pps97/assignments/projects
 http://www.invitrogen.com