Polymerase Chain Reaction

What is PCR?

PCR is an exponentially progressing

synthesis of the defined target DNA

sequences in vitro.
 What is PCR? :
Why “Polymerase”?

It is called “polymerase” because the

only enzyme used in this reaction is
DNA polymerase.
 What is PCR? :
Why “Chain”?

It is called “chain” because the

products of the first reaction become
substrates of the following one, and
so on.
• Kary B Mullis discovered PCR in
1985 and received Nobel price
in 1993 in chemistry.
• PCR highly revolutionized
prenatal diagnosis.
• Very important in forensic
science.
• Potential application in
palaeontology and
archaeology.
 What is PCR? :
The “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
 1. TARGET / TEMPLATE DNA

 This is the DNA/gene that you wish to amplify. The default concentration of
DNA used in the labortatory is generally is 1ng μl-1 of PCR reaction. However
this concentration can vary by a few orders of magnitude depending on the
target gene concentration and source of DNA.

 Higher amounts of template DNA can increase the yield of non specific PCR
products, but if the fidelityof the reaction is crucial, one should limit both
template DNA amounts and number of PCR cycles.

 Although several reagents used in DNA extraction and purification protocols

such as phenol, EDTA and Proteinase K can inhibit the enzyme.
Isopropanol precipitation of NA and treatment of DNA pellets with 70%
ethanol is usually effective in removing traces of contaminants from the DNA
2.Good primer design is essential for successful reactions
 Primer Length:
It is generally accepted that the optimal length of PCR primers
is 18-22 bp.
This length is long enough for adequate specificity and short
enough for primers to bind easily to the template at the
annealing temperature
 Primer Melting Temperature

Primer Melting Temperature (Tm) by definition is the

temperature at which one half of the DNA duplex will
dissociate to become single stranded and indicates the
duplex stability.
Primers with melting temperatures in the range of 52-58
o
C generally produce the best results.
Primers with melting temperatures above 65oC have a
tendency for secondary annealing.
Tm = (4(G+C)+2(T+A))
 Primer Annealing Temperature

The primer melting temperature is the estimate of the DNA-

DNA hybrid stability and critical in determining the annealing
temperature.

Too high Ta will produce insufficient primer-template

hybridization resulting in low PCR product yield.

Too low Ta may possibly lead to non-specific products

caused by a high number of base pair mismatches.

Mismatch tolerance is found to have the strongest influence

on PCR specificity.
PRIMER QUEST
is designed to take an input sequence (cut and paste) in FASTA format and search
that sequence for optimal primer sets using a set of optimizing parametes.

The cardinal parameters are primer length, GC content (%), and Tm. Defaults are
24nt, 50%GC, and a Tm of 60°C. Amplicon size will vary according to the primer
pairs selected.

The logic of Primer quest involves selecting all possible forward and reverse
primers within the input sequence and then, prioritizing based upon all of the design
considerations discussed .
 3. Deoxynucleotide triphosphates (dNTPs)

These are the nucleotide bases added to the growing DNA strand by
the DNA polymerase.The concentration of each dNTP in the reaction
mixture is usually 200μM.

It is very important to have equal concentrations of each dNTP(dATP,

dTTP,dCTP, dGTP), as inaccuracy in the concentration of even a
single dNTP dramaticaly increases the misincorporation level.
 4. Enzyme : polymerase
Initially klenow polymerase was used.
A breakthrough came with the introduction of Taq DNA polymerase
(Lawyer et al. 1989)
Taq DNA Polymerase however lack a proofreading activity(3’-5’)
Other polymerase used are…..
 5. Magnesium

Magnesium ion concentration can affect the specifity and efficiency of the
reaction and is therefore identified as a critical component.

Almost all polymerases require divalent cations for their activity but some
tend to function in buffers containing Mn2+ as well, although a little less
efficiently. It is a required cofactor for thermostable DNA polymerases.

The polymerase enzyme functions actively in higher concentrations of Mg2+

and has lower fidelity i.e. the enzyme is more error prone at excess
concentration of the Mg2+ ions. Magnesium ion concentration can be lowered,
so that the polymerases are barely processive. This will result in higher fidelity
of amplification.
Mg2+ in the PCR mixture stabilizes dsDNA and raises the Tm. Mg2+
concentration therefore is an important for controlling the specificity of the
reaction. A low Mg2+ concentration requires more stringent base pairing in the
annealing step.

dNTPs and oligonucleotides bind to Mg2+ and therefore its concentration

should exceed the molar concentration of phosphate groups that are
contributed from both the dNTPs and primers. dNTPs bind in equimolar
concentration with Mg2+.

The concentration of Mg2+ is dependent on the empirical proportions of

template and primer DNA and will need to be standardized for an experiment.
Polymerase activity when plotted against the Mg2+ gives a bell curve, and the
.
highest activity is seen at 1.2 to 1.3mM. Standard PCR buffers contain 1.5mM of
MgCl2.
Effect of Mg conc. On Polymerase Chain Reaction
 6. Buffers

Buffers are necessary to create optimal conditions for activity of enzyme.

Buffers often contain Tris-HCL, KCl.

The KCl salt in the PCR buffer acts by neutralizing the charge present on the
backbone of DNA. During the elongation step of the PCR, the primer has to
anneal or stick properly to the template and this is facilitated by the KCl.
Thus, it functions by reducing the repulsion between the negatively charged
DNA strands i.e. the primer and template, thereby stabilizing the primer-
template binding.

The temperature that is used during the annealing step of the PCR is less
definitive and the salt helps in the annealing of the primer so the polymerase
can start adding nucleotides from the bound primer properly.
The second generation of PCR buffers has the combinatorial effect of
KCl and (NH4)2SO4.

The K+ ions bind to the phosphate backbone where as the NH4+ exists
both in its ionic form and as ammonia. It interacts with by means of
hydrogen bonds. These interactions can help destabilize the weak
hydrogen bonds between mismatched bases during annealing.

This combined role helps in specificity of primer annealing in a wide

range of temperatures.
 Steps in
PCR

• Denaturation
• Annealing
• Extension
 Denaturation
• High temperature treatment (95c).
• Duration of this step is 1-2mins
• Melt of ds DNA
• Tm: melting temperature
• Consequences of DNA Strands Separation
• Decrease in hydrophobic interactions between DNA bases
• Increase in UV absorbance
 Annealing
• PCR involves two primers that flank the DNA sequence that
has to be amplified.
• The primers hybridize to the opposite strands of DNA .
• And orient the DNA which facilitates the DNA synthesis by
polymerase.
• Optimum annealing temperature is approximately 54c.

EXTENSION
• Optimum temp is 72c for 1min
 Cycling

Cycle number
Ramp time
 Factors affecting PCR
1. DIVALENT CATION

• Magnesium is required as a co-factor for thermostable DNA polymerase.

• Taq polymerase is a magnesium-dependent enzyme and determining the optimum

concentration to use is critical to the success of the PCR reaction.

• Some of the components of the reaction mixture such as template concentration,

dNTPs and the presence of chelating agents (EDTA) or proteins can reduce the

amount of free magnesium present thus reducing the activity of the enzyme.

• Primers which bind to incorrect template sites are stabilized in the presence of

excessive magnesium concentrations and so results in decreased specificity of the

reaction.

• Excessive magnesium concentrations also stabilize double stranded DNA and prevent

complete denaturation of the DNA during PCR reducing the product yield.
2. SIZE OF THE DNA TEMPLATE

PCR works readily with a DNA template of up to two to three thousand base pairs in
length.
However, above this size, product yields often decrease, as with increasing length
stochastic effects such as premature termination by the polymerase begin to
affect the efficiency of the PCR.

3. Polymerase errors

• Taq polymerase lacks a 3' to 5' exonuclease activity.

• The lack in 3' to 5' proofreading of the Taq enzyme results in a high error rate of
approximately 1 in 10,000 bases, which affects the fidelity of the PCR.
• Especially if errors occur early in the PCR with low amounts of starting material,
causing accumulation of a large proportion of amplified DNA with incorrect
sequence in the final product.
• Some of the high fidelity polymerases are Tma from Thermococcus litoralis,Pfu
Pyrococcus furiosus
 4. dNTPs

• Deoxynucleotides (dNTPs) may bind Mg2+ ions and thus

affect the concentration of free magnesium ions in the
reaction.
• In addition, excessive amounts of dNTPs can increase the
error rate of DNA polymerase and even inhibit the reaction.
• An imbalance in the proportion of the four dNTPs can result
in misincorporation into the newly formed DNA strand and
contribute to a decrease in the fidelity of DNA polymerase
5. Cycle Settings
• Reaction cycle lengths, temperatures, and number of cycles,
all have a critical role in determining how well a PCR will
work.

• The initial heating step must be long enough to completely

denature the template and cycles must be long enough to
prevent melted DNA from reannealing to itself.

• Increased yield can be achieved by increasing the extension

time about every 20 cycles, to compensate for less enzyme to
amplify more template.
Other factors are:

Addition of certain proteins, such as, bovine serum albumin enhances

PCR efficiency by protecting the DNA polymerase and by binding to PCR
inhibitors.
Certain compounds act as inhibitors of PCR. Presence of such a
compound in the reaction mixture would, therefore, have an adverse effect
on PCR efficiency. For eg. Inhibitors may be present in the original sample,
such as blood, fabrics, tissues and soil but may also be added as a result
of the sample processing and DNA extraction techniques used.[Excess
salts including KCl and NaCl, ionic detergents such as sodium
deocycholate, sarkosyl and SDS, ethanol, isopropanol and phenol among
others, all contribute via various inhibitory mechanisms, to the reduction of
PCR efficiency.[
 LIMITATION
• Amplicon size (200–1000 bp)
• Sensitive to inhibitors
• Contamination
• Error rate during amplification