Polymerase Chain Reaction (PCR) is a technique that amplifies a specific DNA sequence. It involves denaturing DNA, annealing primers to the DNA, and extending the primers to replicate the DNA. Through repeated cycles of heating and cooling, the targeted DNA sequence is exponentially amplified, allowing it to be detected and manipulated. PCR is used to identify infectious organisms like viruses, detect genetic mutations, and replicate DNA for further analysis. Careful primer design is important for PCR to specifically amplify the desired DNA sequence.

1. Polymerase Chain Reaction

2.  An in-vitro technique that takes specific sequence of DNA of
small amount and amplifies it to be used for further testing.
 Able to a identify and manipulate DNA,
 detect infectious organisms, including the viruses that cause
AIDS, hepatitis, tuberculosis
 Detect genetic variations, including mutations in human
genes
 Purpose:To amplify a lot of double-stranded DNA molecules
(fragments) with same (identical) size and sequence by
enzymatic method and cycling condition.

3. STEPS
I. Denaturation

4. II. Annealing
 Primers bind to their complementary sequences
 The primers dictate the part of the template that will be
amplified
 Temp: 50-70C (dependant on the melting temperature of the
expected duplex)

5. III. Primer extension
 DNA synthesis occurs (68–72C)
 Polymerase synthesizes a copy of the template DNA by
adding nucleotides to the hybridized primers.
 DNA polymerase replicates the template DNA by
simultaneously extending the primers on both strands of
the template

6.  Cycling
 3 Steps: One cycle  one copy of double-stranded DNA
has been replicated into two copies.
 DNA 1 copy
 After PCR

7. Components of PCR Reaction

8. Primers
 Critical component which determine the specificity of the PCR
 Primers are designed to have a sequence which is the reverse
compliment a region of template DNA to which we wish the
primer to anneal
 They are single- stranded DNA fragments, usually 20–30 bases in
length.
 The forward primer must bind to the target DNA sequence just 5′
to the sequences intended to be amplified.
 The reverse primer  5′ to the sequence to be amplified opposite
strand of the DNA.

9. PCR Primer Design Guidelines
 Primer Length: (proportional to annealing efficiency)
 Should be 17-28 bases in length.
 The longer the primer, the smaller the fraction of primed templates and
more inefficient the annealing
 Te shorter the primer, the more quickly it will anneal to target DNA
 Care has to be taken that the primers are not complementary to each
other, particularly at their 3' ends
 Primer Secondary Structures:
 Produced by intermolecular or intramolecular interactions affects the
yield of the product
Hairpins Cross Dimer

10.  Primer melting temperature (Tm)
 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°C
should be preferred (increase the probability of primer
binding)
 temp > 60°C  Chances of secondary annealing

11.  The GC content of the sequence gives a fair indication of the
primerTm
 The formula for primerTm calculation:
Tm = 4(G + C) + 2(A +T)=°C
 [Base composition should be 50-60% (G+C)]
 Presence of G or C bases within the last five bases from the 3'
end, known as the GC clamp
 Promote specific binding at the 3' end due to the stronger bonding
of G and C bases.
 More than 3 G's or C's should be avoided in the last 5 bases at
the 3' end of the primer (promote mispriming)

12.  Primer AnnealingTemperature:
 Determines the DNA-DNA hybrid stablity
 typically lower than theTm by approximately 5°C to 10°C
 Too highTa will produce insufficient primer-template
hybridization resulting in low P
 Too lowTa may possibly lead to non-specific products caused by a
high number of base pair mismatches CR product yield
 Repeats:
 di-nucleotide occurring many times consecutively and should be
avoided (Max is 4)  leads misprime
 Runs:
 Primers with long runs of a single base should generally be avoided
(AGCGGGGGATGGGG)
 3' End Stability:

13. Parameters for Primer Pair Design
 Amplicon Length:
 Product length = (Position of antisense primer-Position of sense
primer) + 1.
 Product Position:
 Sequence close to the 3' end is known with greater confidence
 Optimum AnnealingTemperature