Procedure used to amplify target DNA sequence or gene of interest.

1. POLYMERASE CHAIN
REACTION (PCR)

2. INTRODUCTION:
• Polymerase chain reaction as the name suggests it a continuous chain reaction in
which enzyme polymerase stitches nucleotides i.e dNTPs one after other to form
DNA. It is an amplification process.
• It is a revolutionary method developed by Kary Mullis in the 1983.
• Process of making many copies from target DNA in a thermal cycler machine.
• Mechanism is similar to DNA replication but since this is in- vitro (PCR reaction
chamber), polymerase enzyme is required for polymerization.

3. PRINCIPLE:
The double-stranded DNA of interest is denatured to separate
into 2 individual strands.
Each strand is allowed to hybridize with a primer
(renaturation).
 The primer-template duplex is used for DNA synthesis (DNA
polymerase).
Denaturation, renaturation & synthesis are repeated again &
again to generate multiple forms of target DNA.

4. COMPONENTS OF PCR:
i. Template
 It is target DNA sequence to be amplified.
ii. Primers
 Oligonucleotides used for priming, should be atleast 16 nts and preferably 20-24 nts in length.
 They are designed to anneal on opposite strands of the target sequence so that they will be
extended towards each other by addition of nucleotides to their 3’ ends.
 If the DNA sequence being amplified is known, then primer design is relatively easy.
iii. dNTPs
 The 4 dNTPs, dATP, dGTP, dCTP and dTTP, used at saturating concentration (200 m M each).

5. iv. Enzymes
 Thermostable DNA polymerases from a number of thermophilic bacteria are used for PCR.
 The most common is Taq polymerase from Thermus aquaticus. It survives the denaturation step of
95ºC for 1-2 min, having a half-life of more than 2hr at this temperature.
 It carries a 5’-3’ polymerization dependant exonuclease activity, but lack in 3’-5’ exonuclease
activity (proof reading).
 Hence, it is more prone for introducing errors. There are high-fiedality thermostable enzymes with
3’-5’ exonuclease activity. e.g., Vent polymerase, pfu polymerase.
v. Buffer
 The standard buffer for PCR contains 50 mM KCl, 10 mM Tris.Cl and 1.5 mM MgCl2. pH is
approximately 7.2. The presence of divalent cations is critical (Mg2+).

6. STAGES OF PCR CYCLE:
 PCR involves a repetitive series of temperature cycles. Each reaction cycle comprises
of three stages
i. Denaturation (95°C for 1 min)
ii. Primer annealing (55°C for 45 sec)
iii. Extension (72°C for 2 min)
 In the first cycle, the target DNA is separated into two strands by heating to 95ºC-
denaturation.
 The temperature is reduced to around 55ºC to allow the primers to anneal. The actual
temperature depends on the primer lengths and sequences- primer annealing.
 After annealing, the temperature is increased to 72ºC for optimal polymerization which
uses up dNTPs in the reaction mix and requires Mg2+ ion.
 If PCR was 100% efficient, one target molecule would become 2n after ‘n’ cycles. In
practice, 20- 40 cycles are commonly used.
Theoretical yield = 2n ie. cycle 1 = 2, cycle 2 = 4, cycle 3 = 8 copies etc.

8. PCR Components Troubleshooting
i. Buffer
 Most buffers have only KCl (50mM) and Tris (10mM).
 KCl facilitates primer binding but concentrations higher than 50mM inhibit Taq.
ii. MgCl2: required for primer binding
 MgCl2 affects primer DNA binding, product- and primer-template associations,
product specificity, enzyme activity.
 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 and too little magnesium gives
reduced yield.
iii. Primer Designing Criteria
 Ensure that the primers are specific to the target of interest and length 18-30
nucleotides.
 Use online primer design tools when appropriate.

9.  Verify that the primers are complementary to the correct strands of the target
DNA
 GC content not more than 40-60% (removal difficult), if less (no proper binding).
 3’ end is very critical- new strand extends from here.
 GC clamp (G or C at 3’ terminus)- as it will ensure proper binding and initialization
of process.
 Inner self complementarity: primers may make intra strand bonds eg. hairpin.
iv. Insufficient quantity of DNA polymerase:
 Review recommendations on the amount of DNA polymerase to use in PCR, and
optimize as necessary.
 Increase the amount of DNA polymerase if the reaction mixture contains a high
concentration of an additive (e.g., DMSO, formamide) or inhibitors from the sample
sources.

10. v. Excess PCR additives or solvents:
 Review the recommended concentrations of PCR additives or co-
solvents. Use the lowest possible concentration when appropriate.
 Adjust the annealing temperatures, as high concentrations of PCR
additives or co-solvents weaken primer binding to the target.
 Increase the amount of DNA polymerase, or use DNA polymerases
with high processivity.
vi. Nonhomogenous reagents:
 Mix the reagent stocks and prepared reactions thoroughly to eliminate
density gradients that may have formed during storage and setup.

11. Thermal Cycling Troubleshooting
i. Suboptimal denaturation:
 Some Taq polymerases require initial denaturation (hot start).
 Optimize the DNA denaturation time and temperature.
 Short denaturing times and low temperatures may not separate double-
stranded DNA templates well. Also, long denaturation times and high
temperatures may reduce enzyme activity.
ii. Suboptimal annealing:
 Annealing temperature: ∼5°C less than Tm of primers and DNA hybrid
(will cause improper binding of both).
 Decrease in annealing temperature result in non-specific binding.
 Increase in annealing temperature result in reduced yield.

12. iii. Suboptimal Extension:
 Select an extension time suitable for the amplicon length.
 Reduce the extension temperature (e.g., to 68°C) to keep the enzyme active during
amplification of long targets (e.g., >10 kb).
 Use DNA polymerases with high processivity for robust amplification even with
short extension times.
iv. Suboptimal number of PCR cycles:
 Adjust the number of cycles (generally to 25–35 cycles) to produce an adequate
yield of PCR products.
 Extend the number of cycles to 40 if DNA input is fewer than 10 copies.
 Half-life of Taq is 30 minutes at 95ºC .
 Therefore if used more than 30 cycles at denaturation times of 1 minute, the Taq
will not be very efficient at this point (as from very beginning it is present in PCR
mix).