POLYMERASE CHAIN REACTION

Aim:-
To perform amplification of target genes in template plasmid DNA using PCR technique.
Introduction:
Kary Mullis in the early 1980s developed a technique which enabled scientists to make
millions of copies of DNA molecule in a very short time. Since then PCR is being employed
as a rapid and inexpensive way of amplifying specific DNA fragments from minute quantities
of template DNA. In 1993, he got Nobel Prize in Chemistry for his dedicated work on PCR.
There are numerous applications of PCR varying from DNA sequencing, in-vitro
mutagenesis, mutation detection, cloning of cDNA and genomic DNA etc.

Fig 1 :- Image showing a PCR machine

Principle:-

PCR utilizes short, user defined DNA sequences called oligonucleotide primers, the
sequence of which are complementary to target regions of genes Because DNA polymerase
can add a nucleotide only onto a pre-existing strand with free 3'-OH group, it uses the primer
which serves as template to which additional nucleotides are sequentially added. The
enzyme DNA polymerase used in PCR adds nucleotides to the pre-existing 3'-OH group and
thus synthesizes new strands of DNA complimentary to the template DNA.
The products of first round of PCR will be two daughter strands which then act as
template for the next round of primer driven DNA synthesis. Further the PCR proceeds for a
definite number of PCR cycles with copies of target sequence doubling during each cycle
until the concentration of reagents become limiting.
Since each cycle of PCR involves creating two new double stranded DNAs from each DNA
molecule present, the amount of DNA theoretically doubles with every cycle of PCR.
Therefore, after two cycles the concentration of DNA increases by 22-fold, after 3 cycles a
23 fold increase, etc. After N cycles, PCR generates a 2N-fold increase in the target DNA.
Finally at the end of the PCR reaction, the specific sequence will be accumulated in billions
of copies (amplicons). Most PCR methods amplify DNA fragments of between 0.1-10kb in
length. 

Fig 2:- Exponential amplification following each cycle.

Materials & Methods

 Template DNA (genomic, plasmid, bacterial colony, etc.)
 Primers (resuspended in sterile water or TE )
 Buffer (usually 10X, usually sold with Taq polymerase )
 MgCl2 (available in 25mM or 50 mM stocks)
 Taq DNA polymerase
 dNTPs (10mM stock)
 Sterile, nuclease-free water
 Gloves
 PCR thermalcycler
 Pipettes (1-10 µl, 5-50 µl, 20-200 µl, and 100-1000 µl)
 Aerosol barrier pipette tips
 PCR tubes (0.2 ml or 0.5 ml)
 Master mix tubes (1.5 ml microcentrifuge tubes)

PCR Components:-
Following are the basic components for a PCR reaction.

1. DNA template: - The sample DNA used for PCR which harbors the target sequence to
amplify. This could be single or double stranded,either plasmid or genomic DNA template.

Template DNA Amount

genomic 1ng-1ug
Plasmid or viral 1pg-10ng

2. Thermostable DNA polymerase:- These are heat stable enzymes which catalyse
template dependent synthesis of DNA. There are variety of DNA polymerses but for routine
PCR, Taq polymerse (0.5–2.5 units per standard 25–50-μL reaction) originally isolated from
Thermus aquaticus which lives in hot springs and is heat stable is the common choice.

3. Oligonucleotide Primers:- Short pieces of single stranded DNA that are complementary
to the target sequence. Oligonucleotide primers are generally 15-30 nucleotides in length
that are complementary to DNA sequences that flank the target region of interest. The
purpose of PCR primers is to provide a “free” 3’-OH group to which the DNA polymerase can
add nucleotides.

An ideal primer should have the following characteristics:-

 They should ideally have a G+C content of 40-60% .Standard reactions contain
typically 0.05–1μM of each primer.
 More than three G or C nucleotides at the 3'-end of the primer should be avoided, as
nonspecific priming may occur.
 The primer should not be self-complementary or complementary to any other primer
in the reaction mixture, in order to avoid primer-dimer and hairpin formation.
 All possible sites of complementarity between primers and the template DNA should
be noted.
 The melting temperature of flanking primers should not differ by more than
5°C.Therefore, the GC content and length must be chosen accordingly.If the primer
is shorter than 25 nucleotides, the approx. melting temperature (Tm) is calculated
using the following formula:,

Tm = 4(G+C) + 2(A+T)

where G, C, A, and T, are the number of respective nucleotides in the primer. If the
primer is longer than 25 nucleotides, the melting temperature should be
calculated using specialized computer programs where the interactions of
adjacent bases, the influence of salt concentration, etc. are evaluated.
 The PCR annealing temperature (Ta) should be approximately 5°C lower than
the primer melting temperature.

4. Nucleotides (dNTPs or deoxynucleotide triphosphates):- Standard PCRs contain

equimolar amounts of dATP, dTTP, dCTP, and dGTP. Concentrations of 200–250 μM of
each dNTP are recommended for Taq polymerase in reactions containing 1.5 mM MgCl2.

5. PCR buffer :- This is included inorder to maintain pH .Tris-Cl, adjusted to a pH between

8.3 and 8.8 at room temperature, is included in standard PCRs at a concentration as low as
10 mM or as high as 66 mM. When incubated at 72°C (the temperature commonly used for
the extension phase of PCR), the pH of the reaction mixture drops by more than a full unit,
producing a buffer whose pH is 7.2.

6. Monovalent cations. Standard PCR buffer contains 50 mM KCl and works well for
amplification of segments of DNA >500 bp in length.

7. Divalent cations:- All thermostable DNA polymerases require free divalent cations—
usually Mg2+—for activity. Magnesium ions have two functions in PCR: reacting with dNTPs
to form complexes that are the substrates for Taq polymerase, and stabilizing the primer–
template complexes. The routinely used concentration of Mg2+ is 1.5mM.
 Stages of PCR

PCR is an iterative process, consisting of three stages. It uses temperature cycling to initiate
and end series of enzyme-catalysed DNA synthesis. The 3 stages in an ideal PCR reaction
include:-

 Denaturation of the template by high temperature (strand separation),

 Annealing of the oligonucleotide primers to the single-stranded target sequence(s),
and
 Extension of the annealed primers by a thermostable DNA polymerase.

Fig 3:- Stages in a PCR cycle

Stages (explained)

1. Denaturation of template DNA: 

An initial denaturation of 30 seconds at 90-95°C is sufficient for most amplicons from pure
DNA templates. For difficult templates such as GC-rich sequences, a longer initial
denaturation of 2–4 minutes at 95°C is recommended prior to PCR cycling to fully
 denature the template. During thermocycling a 15–30 second denaturation at 95°C is
recommended.

2. Annealing of oligonucleotide primers to the denatured template DNA: 

The primers usually 20-25nt in length are designed using prior knowledge of the DNA
sequence of the template. The two primers (forward & reverse) are complementary to
sequences on opposite strands of target DNA. Annealing temperature is based on the
Tm (melting temperature of the primer pair and is typically 45–60°C. Annealing
temperatures is ideally optimized by doing a temperature gradient PCR which is 5°C
below the calculated Tm.

3. Extension: 

The recommended extension temperature is 68-72°C. Extension times are generally 1

minute per kb. A final extension of 5 minutes at 68-72°C is recommended.

4. Cycle number: 
The number of cycles for amplification depends on the number of copies of template
DNA present at the beginning of the reaction and the efficiency of primer extension and
amplification. Generally, 25–35 cycles yields sufficient product.

PCR Reaction Protocol: 

Points to note while performing PCR:-

PCR is very sensitive to contamination from non-target DNA. Several steps should be taken
to reduce the chance for contamination, which are as follows:
• Fresh gloves should be worn for DNA purification and reaction set-up.
• Limit the amount of close contact with sample tubes and reagents.
• DNA sample preparation, reaction mixture assembly and the PCR process, in addition to
the subsequent reaction product analysis, should be performed in sterile area wiped with
70% ethanol.
• The reagents for PCR should be prepared separately and used solely for this purpose.
• Only nuclease-free water should be used in the preparation and suspension of PCR
reagents. • Unless the solution is purchased sterile, autoclaving of all solutions, except
dNTPs, primers and Taq DNA polymerase is recommended.
• Aliquots of PCR reagents should be stored separately from DNA samples.
• A control reaction, omitting template DNA, should always be performed, to confirm the
absence of contamination.

All reaction has to be performed on ice.

Component 10ul reaction Final concentration

10X standard TAQ 2 µl

reaction buffer with
MgCl2

10Mm dNTPs 0.2 µl

10µM forward primer 0.5 µl

10µM reverse primer 0.5 µl

Template DNA 1 µl
 (plasmid/genomic)

Taq DNA 0.20 µl

polymerase

Nuclease free water to 10µl

Notes: Gently mix the reaction. Collect all liquid to the bottom of the tube by a quick spin if
necessary .Make sure to remove air bubbles.

Transfer PCR tubes from ice to a PCR machine with the block preheated to 95°C and begin
thermo cycling. (32 cycles)

Thermocycling conditions

Step Temperature Time

Initial denaturation 95 degree C 2 min
Denaturation 95 degree C 15 sec
Annealing 56 degree C 15 sec
Extension 72 degree C 15 sec
Final extension 72 degree C 5 min
Hold 4 -10 degree C

Visualization of PCR products

The PCR amplified fragments can be run in a 2.5 % Agarose gel containing the
fluorescent DNA intercalating dye Ethidium bromide and DNA bands can then be visualized
in a Gel documentation system with attached UV light or a UV transilluminator.

Results & Discussion