POLYMERASE CHAIN REACTION

Widely used for diagnostic and research purposes

Allows millions to billions copies of a specific DNA

product to be made in vitro

The key enzyme in the reaction is DNA Polymerase;

therefore it was named Polymerase Chain
Reaction

The Polymerase Chain Reaction (PCR) was not a

discovery, but rather an invention
POLYMERASE CHAIN REACTION
APPLICATIONS
Gene cloning
Pathogen identification and diagnosis
Drug discovery
Gene expression studies
Gene sequencing
Disease identification
Genotyping
PCR: 3 Basic Steps
POLYMERASE CHAIN REACTION
BASIC STEPS
1. Denaturation: Heated above the
melting point of the two
complementary strands of the
template DNA, which allows the
strands to separate.
2. Annealing: The temperature is
lowered which allows the primers to
bind to the specific and
complementary DNA sequence to be
amplified
3. Extension: The DNA polymerase is
able to extend the primers by adding
nucleotides to the developing DNA
strand.
PCR REAGENTS
1X Buffer
10mM Tris-HCl, 50mM KCl
MgCl2
1mM - 4mM (1.5mM)
dNTPs
200M
Primers
100nM-1M, 200nM (or less) for real time analysis
DNA polymerase
Taq DNA polymerase is thermostable
1-4 Units (1 unit)
DNA
10pg-1g (20ng)
Initially PCR used the Klenow fragment of E. coli DNA
polymerase - inactivated by high temperatures
Kleppe, Ohtsuka, Kleppe, Molineux, Khorana. 1971. J. Mol. Biol. 56:341.

Required a thermostable DNA

polymerase - Taq DNA polymerase
from Thermus aquaticus, a
thermophilic microorganism
isolated from a hot spring in
Yellowstone National Park
COMMERCIAL THERMOSTABLE POLYMERASES
PCR - BEFORE THE THERMOCYCLER

95 C 55 C 72 C
5 min 3 min 5 min

35 times

8 BORING hours per PCR!

THERMAL CYCLER
The device has a thermal block with holes
where tubes holding the reaction
mixtures can be inserted. The cycler then
raises and lowers the temperature of the
block in discrete, pre-programmed steps.

Modern PCR machines uses Peltier

element to prevent evaporation. Quality
thermal cyclers often contain silver blocks
to achieve fast temperature changes and
uniform temperature throughout the
block. Other cyclers have multiple blocks
with high heat capacity, each of which is
kept at a constant temperature, and the
reaction tubes are moved between them
by means of an automated process
A simple thermocycling protocol
1X 35X 1X

94C 94C

3 min 1 min 72C

1 min
55C
Initial denaturation
of DNA 45 sec

denaturation

extension
4C

annealing
hold
PCR Problems
Taq is inactive at low temperatures
At low temperatures mis-priming is likely

TEMP EXTENSION RATE

55o C 24 nt/sec

37o C 1.5 nt/sec

22o C 0.25 nt/sec 150 nucleotides in 10 min

 Cures for mis-priming
Cheap fixes
Set up reactions on ice

Hot-start PCR holding one or more of the PCR components until the first
heat denaturation
Manually - delay adding polymerase
Wax beads
Polymerase antibodies

Touch-down PCR set stringency of initial annealing temperature high,

incrementally lower with continued cycling

PCR additives
0.5% Tween 20
5% polyethylene glycol 400
betaine
DMSO
PRIMER DESIGN
Typically 20 to 30 bases in length
Annealing temperature dependent upon
primer sequence (~ 50% GC content)
Avoid secondary structure, particularly 3
Avoid primer complementarity (primer
dimer)
The last 3 nucleotides at the 3` end is the
substrate for DNA polymerase - G or C
Many good freeware programs available
PRIMER DESIGN SOFTWARE

Many free programs available online

OLIGO
PRIMER
PrimerQuest
PRIMER DIMERS
PAIR OF PRIMERS
5-ACGGATACGTTACGCTGAT-3(Primer 1)

5-TCCAGATGTACCTTATCAG-3(Primer 2)

COMPLEMENTARITY OF PRIMER 3 ENDS

5-ACGGATACGTTACGCTGAT-3
3-GACTATTCCATGTAGACCT-5

RESULTS IN PCR PRODUCT

Primer 1
5-ACGGATACGTTACGCTGATAAGGTACATCTGGA-3
3-TGCCTATGCAATGCGACTATTCCATGTAGACCT-5
Primer 2
RULES OF THUMB FOR PCR CONDITIONS
Add an extra 3-5 minute (longer for Hot-start Taq) to your
cycle profile to ensure everything is denatured prior to
starting the PCR reaction

Approximate melting temperature

If GC content is < 50% start 5oC beneath Tm for
annealing temperature
If GC content 50% start at Tm for annealing
temperature

Extension @ 72oC: rule of thumb is ~500 nucleotide per

minute. Use 3 minutes as an upper limit without special
enzymes
COMMON PCR ADDITIVES
BSA (usually at 0.1 to 0.8 g/L final concentration)
Stabilize Taq polymerase & overcome PCR inhibitors

DMSO (usually at 2-5% v/v, inhibitory at 10% v/v)

Denaturant - good at keeping GC rich template/primer strands from forming
secondary structures.

Glycerol (usually at 5-10% v/v)

Increases apparent concentration of primer/template mix, and often increases
PCR efficiency at high temperatures.

Stringency enhancers (Formamide, Betaine, TMAC)

Concentrations used vary by type
Enhances yield and reduces non-specific priming

Non-ionic detergents (Triton X, Tween 20 or Nonidet P-40) (0.11%)

NOT SDS (0.01% SDS cuts Taq activity to ~10% of normal)
Stabilize Taq polymerase & suppress formation of 2 structure
PCR ADDITIVES - LITERATURE
ADDITIVE REFERENCES
Amplifications 5: 16
DMSO Gene 140: 1
(dimethyl sulfoxide) Nucleic Acids Research 18: 1666

Biochemistry 32: 137

BioTechniques 21: 1102
Betaine Genome Research 6: 633
(N,N,N-trimethylglycine Nucleic Acids Research 25: 3957
= [carboxymethyl] trimethylammonium) Proceedings of the National Academy of Sciences of the United States of America 70: 298
Trends in Biochemical Science 22: 225

Formamide Nucleic Acids Research 18: 7465

Non-ionic detergents Biotechniques 12: 332

e.g. Triton X-100, Tween 20 or Nonidet P-40 (NP-40) Nucleic Acids Research 18: 1309

TMAC Nucleic Acids Research 18: 4953

(tetramethylammonium chloride) Nucleic Acids Research 23: 3343

dC7GTP Nucleic Acids Research 16: 3360

(7-deaza-2'-deoxyguanosine)

Applied and environmental microbiology 62:1102-1106

BSA BioTechniques 23:504
(bovine serum albumin) BioTechniques 25:564
Nucleic Acids Research 16: 9775
TYPICAL PCR TEMPS/TIMES
Initial 90o 95o C 1 3 min
denaturation

Denature 90o 95o C 0.5 1

min
25 40
Primer 45o 65o C 0.5 1
cycles
annealing min
Primer 70o 75o C 0.5 2
extension min

Final extension 70o 75o C 5 10

min
Stop reaction 4o C or 10 mM EDTA hold
EXTENSION - THE REPLICATION FORK
3 5
5 3
3 5 3 5 Primase
Single strand
LAGGING STRAND binding
5 proteins
Okazaki 5
fragment 3
5
RNA
Primers
DNA
Polymerase
5
3
Helicase

LEADING STRAND
5
3
PCR V/s NATURAL REPLICATION

FUNCTION PCR REPLICATION

MELTING DNA Heat Helicase
SSBP
Topoisomerase
POLYMERIZING DNA Taq/Pfu DNA Pol
PROVIDING PRIMER Added to the Primase
reaction mixture
manually
JOINING NICKS N/A Ligase
 100
Melting
94 oC
Temperature
50

0
Time

3 5
5 3
 100
Melting
94 oC
Temperature
50

0
Time

3 5

HEAT

5 3
 100
Melting Melting
94 oC Extension 94 oC
Annealing
Temperature Primers 72 oC
50
50 oC

0
Time

3 5
5

5
5 3
 30x

100
Melting Melting
94 oC Extension 94 oC
Temperature
Annealing
Primers 72 oC
50
50 oC

0
Time

3 5

HEAT
5

5
HEAT

5
5 3
 30x

100
Melting Melting
94 oC Extension 94 oC
Temperature
Annealing
Primers 72 oC
50
50 oC

0
Time

3 5
5

5
5

5
5

5
5 3
 30x

100
Melting Melting
94 oC Extension 94 oC
Temperature
Annealing
Primers 72 oC
50
50 oC

0
Time
3 5
5

5
5
5 3

HEAT
5

5
HEAT

5
 30x

100
Melting Melting
94 oC Extension 94 oC
Temperature
Annealing
Primers 72 oC
50
50 oC

0
Time
3 5
5

5
5 5
5 3

5
5

5
5

5
5
 30x

100
Melting Melting
94 oC Extension 94 oC
Temperature
Annealing
Primers 72 oC
50
50 oC

0
Time
3 5
5

5
5 5
5 3

5
5
Fragments of
defined length
5
5

5
5
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

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

PCR Optimisation 3: Primer Design
Specific to sequence of interest
Length 18-30 nucleotides
Annealing temperature 50oC-70oC
Ideally 58oC-63oC
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
PCR Optimisation 4: Cycling Conditions
Denaturation:
Some Taq polymerases require initial denaturation (hot
start)
Annealing temperature:
~ 5oC less than Tm of primers
Tm = 4(G + C) + 2(A + T)oC (or use of primer software)
Decrease in annealing temperature result in non-specific
binding
Increase in annealing temperature result in reduced yield
PCR Optimisation 5: Cycle Number
25-40 cycles Theoretical yield = 2n
ie. cycle 1 = 2, cycle 2 = 4, cycle 3 = 8, etc
Half-life of Taq is 30
eg. if you start with 100 copies after 30 cycles you
minutes at 95oC will have 107, 374, 182, 400 copies
Therefore if you use
more than 30 cycles
at denaturation
times of 1 minute,
the Taq will not be
very efficient at this
point
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.
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