Polymerase chain reaction , its principles and its medical applications for medical and dental students

1. Dr.S.Sethupathy,M.D.,Ph.D,
Professor of Biochemistry,
RMMC,
AU

2. What is PCR?What is PCR?
It was invented in 1983 by Dr. Kary
Mullis, for which he received the Nobel
Prize in Chemistry in 1993.
PCR is an exponentially progressing
synthesis of the defined target DNA
sequences in vitro.

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

4. What is PCR? :What is PCR? :
Why “Chain”?Why “Chain”?
It is called “chain” because the
products of the first reaction become
substrates of the following one, and
so on.

5. What is PCR? :What is PCR? :
The “Reaction” ComponentsThe “Reaction” Components
1) Target DNA - contains the sequence to be amplified.
2) Pair of Primers - oligonucleotides that define the sequ
to be amplified.
3) dNTPs - deoxynucleotidetriphosphates: DNA building
4) Thermostable DNA Polymerase - enzyme
that catalyzes the reaction
5) Mg++
ions - cofactor of the enzyme
6) Buffer solution – maintains pH and ionic
strength of the reaction solution suitable for
the activity of the enzyme

6. THERMOCYCLERPCR tube

7. Denature (heat to
95o
C)
Lower temperature to
56o
C Anneal with primers
Increase temperature to
72o
C DNA polymerase +
dNTPs

11. DNA copies vs Cycle number
0
500000
1000000
1500000
2000000
2500000
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Cycle number
DNAcopies

12. PCR Reagents
1X Buffer
10mM Tris-HCl, 50mM KCl
MgCl2
1mM - 4mM (1.5mM)
dNTPs
200μM
Primers
100nM-1μM, 200nm (or less) for real time analysis
DNA polymerase
Taq DNA polymerase is thermostable
1-4 Units (1 unit)
DNA
10pg-1μg (20ng)

13. Applications of PCRApplications of PCR
Classification
of organisms
Genotyping
Molecular
archaeology
Mutagenesis
Mutation
detection
Sequencing
Cancer research
Detection of
pathogens
DNA
fingerprinting
Drug discovery
Genetic matching
Genetic
engineering
Pre-natal
diagnosis

14. Different types of buffers

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

17. Number of cycles
0 10 15 20 25 30
Size
Marker

18. PCR Optimisation 1: Buffers
Most buffers have only KCl (50mM) and Tris
(10mM)
Concentrations of these can be altered
KCl facilitates primer binding but concentrations
higher than 50mM inhibit Taq
DMSO, BSA, gelatin, glycerol, Tween-20, Nonidet
P-40, Triton X-100 can be added to aid in the PCR
reaction
Enhance specificity, but also can be inhibitory
Pre-mixed buffers are available

19. PCR Optimisation 2: MgCl2
MgCl2: required for primer binding
MgCl2 affects primer binding, Tm of template DNA,
product- and primer-template associations, product
specificity, enzyme activity and fidelity
dNTPs, primers and template chelate and sequester the Mg
ion, therefore concentration should be higher than dNTPs
(as these are the most concentrated)
Excess magnesium gives non-specific binding
Too little magnesium gives reduced yield

20. PCR Optimisation 3: Primer Design
Specific to sequence of interest
Length 18-30 nucleotides
Annealing temperature 50o
C-70o
C
Ideally 58o
C-63o
C
GC content 40-60%
3’ end critical (new strand extends from here)
GC clamp (G or C at 3’ terminus)
Inner self complementarity:
Hairpins <5, dimers <9
3’ complementarity:
<3-4 bases similar to other primer regions

21. PCR Optimisation 4: Cycling Conditions
Denaturation:
Some Taq polymerases require initial denaturation (hot
start)
Annealing temperature:
~ 5o
C less than Tm of primers
Tm = 4(G + C) + 2(A + T)o
C (or use of primer software)
Decrease in annealing temperature result in non-specific
binding
Increase in annealing temperature result in reduced
yield

22. PCR Optimisation 5: Cycle Number
25-40 cycles
Half-life of Taq is
30 minutes at 95o
C
Therefore if you
use more than 30
cycles at
denaturation
times of 1 minute,
the Taq will not be
very efficient at
this point
Theoretical yield = 2n
ie. cycle 1 = 2, cycle 2 = 4, cycle 3 = 8, etc
eg. if you start with 100 copies after 30 cycles
you will have 107, 374, 182, 400 copies

23. In summary
Primer length should not exceed 30 mer.
Tm, not more than 60 degree .
GC Content should be in the range of 40-60 % for optimum
PCR efficiency.
 Primers should end (3′) in a G or C, or CG or GC: this
prevents “breathing” of ends and increases efficiency of
priming.

24. Primer3
http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi

25. Primer Problems
primers should flank the sequence of interest
primer sequences should be unique
primers that match multiple sequences will give multiple products
repeated sequences can be amplified - but only if unique flanking
regions can be found where primers can bind

26.  Sequence Specific Oligonucleotide (SSO) probe
 Amplified fragment-length polymorphism to generate
finger prints
 Large VNTR regions (10-30 b.p. repeat)
 Short Tandem Repeats (STR) (2-7 b.p. repeat)
 RAPD using universal primers
 Rep- PCR (ERIC primers)
 PCR- Ribotyping (16S rDNA regions)
PCR Based Methods

27. Variations of the 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
Real time PCR

28. Types of PCR
Long PCR: Used to amplify DNA over the entire length up to 25kb of genomic DNA
segments cloned.
Nested PCR: Involves two consecutive PCR reactions of 25 cycles. The first PCR uses
primers external to the sequence of interest. The second PCR uses the product of the
first PCR in conjunction with one or more nested primers to amplify the sequence
within the region flanked by the initial set of primers.
Inverse PCR: Used to amplify DNA of unknown sequence that is adjacent to known
DNA sequence.
Quantitative PCR: Product amplification w r t time, which is compared with a
standard DNA.
Hot start PCR: Used to optimize the yield of the desired amplified product in PCR
and simultaneously to suppress nonspecific amplification.

29. 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.

30. 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
DNA Polymerase- Eubacterial type I DNA polymerase, Pfu
These thermophilic DNA polymerases show a very small
polymerase activity at room temperature.

31. Nested PCR
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 (as they lie / are nested
within the first fragment) 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. 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.

33. 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.

34. Long PCR
Extended or longer than standard PCR,
meaning over 5 kilobases (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

35. 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

36. Why real time PCR ?
• QUANTITATION OF mRNA
– northern blotting
– ribonuclease protection assay
– in situ hybridization
– RT-PCR
• most sensitive
• can discriminate closely related mRNAs
• technically simple
• but difficult to get truly quantitative results using
conventional PCR

37. Real-Time PCRReal-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

38. Traditional PCR has advanced from detection at the
end-point of the reaction to detection while the
reaction is occurring (Real-Time).
Real-time PCR uses a fluorescent reporter signal to
measure the amount of amplicon as it is generated.
This kinetic PCR allows for data collection after
each cycle of PCR instead of only at the end of the 20
to 40 cycles.

39.  Real-time PCR advantagesReal-time PCR advantages
 * amplification can be monitored real-time
 * no post-PCR processing of products
 (high throughput, low contamination risk)
 * ultra-rapid cycling (30 minutes to 2 hours)
 * wider dynamic range of up to 1010
-fold
 * requirement of 1000-fold less RNA than conventional
assays
 (6 picogram = one diploid genome equivalent)
 * detection is capable down to a two-fold change
 * confirmation of specific amplification by melting curve
analysis
 * most specific, sensitive and reproducible
 * not much more expensive than conventional PCR
 (except equipment cost)

40. Real-time PCR disadvantagesReal-time PCR disadvantages
* Not ideal for multiplexing
* setting up requires high technical skill and support
* high equipment cost
* intra- and inter-assay variation
* RNA liability
* DNA contamination (in mRNA analysis)

41. Applications of PCRApplications of PCR
Classification
of organisms
Genotyping
Molecular
archaeology
Mutagenesis
Mutation
detection
Sequencing
Cancer research
Detection of
pathogens
DNA
fingerprinting
Drug discovery
Genetic matching
Genetic
engineering
Pre-natal
diagnosis

42. PCR Virtues
High sensitivity
Can detect and quantify specific
events
Higher stability of DNA permits
analysis of food samples.
Quantitative and qualitative

43. Some applications of PCR.
Forensic medicine.
Preimplantation Genetic Diagnosis
(PGD).
Archeology.
Paternity testing.

44. Forensic uses of PCR
PCR can be used to amplify DNA
from a small amount of cells (about
1000 cells).
The amplified DNA from cells can be
used in DNA fingerprinting analysis to
determine who was at the crime scene.

45. DNA fingerprinting using PCR in forensic
investigations.
DNA is isolated from blood at a crime
scene and amplified by PCR.
The amplified DNA is digested with
restriction enzymes and resolved on an
agarose gel.
Southern blot analysis is performed to
give a DNA fingerprint.

46. How reliable is DNA fingerprinting?
DNA regions chosen are ones known to be
highly variable from one person to another.
In most forensic cases, the probability of
two people having identical DNA
fingerprints is between one chance in
100,000 and one in a billion.
The exact number depends on the number
of probes used to different regions of
human chromosomal DNA.

47. Satellite DNA can be used as markers for DNA
fingerprinting.
Satellite DNA consists of tandemly repeated
base sequences within the human genome.
The most useful satellite DNA for forensic
purposes are microsatellites having repeating
units of only a few base pairs, and the number
of repeats are highly variable from one person
to another.
Microsatellite DNA is also called a simple
tandem repeats (STRs).

48. STRs in DNA fingerprinting.
The greater the number of STRs
analyzed in a DNA sample, the more
likely the DNA fingerprint is unique to
an individual.
PCR is used to selectively amplify
particular STRs before electrophoresis.
PCR is especially valuable when DNA is
in poor condition or available in minute
quantities.

49. PCR use in Pre-implantation Genetic
Diagnosis (PGD).
PGD is a way to determine if human
embryos from in vitro fertilization have
genetic defects (for example, cystic fibrosis).
A cell is removed from an eight cell embryo
and the DNA is analyzed by PCR for genetic
defects.
Only healthy embryos are implanted into a
mother’s uterus.
Should this technology be used for things
like gender selection?

50. Thank you