Polymerase chain reaction

PCR for medical 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 sequence
to be amplified.
3) dNTPs - deoxynucleotidetriphosphates: DNA building blocks.
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. THERMOCYCLER
PCR tube

7. Denature (heat to
95
o
C)
Lower temperature to
56
o
C Anneal with primers
Increase temperature to
72
o
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 91011121314151617181920212223
Cycle number
D
N
A

c
o
p
i
e
s

12. PCR Reagents
1X Buffer
10mM Tris-HCl, 50mM KCl
MgCl
2
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. Different types of buffers

15. Fragments of
defined length
PCR
Melting
94
o
C
Melting
94
o
C
Annealing
Primers
50
o
C
Extension
72
o
C
T
e
m
p
e
r
a
t
u
r
e
100
0
50
T i m e
30x
3’5’
5’3’
5’
5’
5’
5’
5’
5’
5’
5’
5’
5’

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

17. 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

18. PCR Optimisation 2: MgCl
2
MgCl
2: required for primer binding
MgCl
2 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

19. PCR Optimisation 3: Primer Design
Specific to sequence of interest
Length 18-30 nucleotides
Annealing temperature 50
o
C-70
o
C
Ideally 58
o
C-63
o
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

20. PCR Optimisation 4: Cycling Conditions
Denaturation:
Some Taq polymerases require initial denaturation (hot
start)
Annealing temperature:
~ 5
o
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

21. PCR Optimisation 5: Cycle Number
25-40 cycles
Half-life of Taq is
30 minutes at 95
o
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 = 2
n
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

22. 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.

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

24. 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

25. Sequence Specific Oligonucleotide (SSO) probe
Amplified fragment-length polymorphism to generate
finger prints
Large VNTR (Variable number tandem repeats) regions
(10-30 b.p. repeat)
Short Tandem Repeats (STR) (2-7 b.p. repeat)
RAPD(random amplified polymorphic DNA) using
universal primers
Rep- PCR( repetitive sequence based PCR) (ERIC-
enterobacterial repetitive intergenic consensus primers)
PCR- Ribotyping (16S rDNA regions)
PCR Based Methods

26. 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

27. 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

28. 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.

29. 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 10
10
-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)

30. 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)

31. 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

32. Thank you