PCR (Polymerase Chain Reaction)

MATERIALS REQUIRED
Primer pair: A primer is a nucleic acid strand, or a related molecule that serves as a starting point for
DNA replication. A primer is required because most DNA polymerases, enzymes that catalyze the
replication of DNA, cannot begin synthesizing a new DNA strand but can only add to an existing strand of
nucleotides.
These are the oligonucleotides, 18-25 bp in length, each complementary to a stretch of DNA (in the
flanking region) to the 3' side of the region to be amplified. G+C content of primers should be between
40-60% with even distribution of all four bases, with no complementarity) within and in between the
primer pair.
Buffer: Tris-Cl buffer adjusts the optimum pH (pH 8.3 to 8.8 at room temperature) for this reaction to
take place. When incubated at 72°C (the temperature 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 which is
optimum for the functioning of Taq Polymerase.
Divalent cations:All thermostable DNA polymerases require free divalent cations – usually Mg2+ for
activity. These are usually provided in the buffer itself.
Taq polymerase: The DNA polymerase used in PCR is derived from Thermus aquaticus which is stable
even at prolonged exposures at 95°C. This removes the requirement of adding the enzyme after every
cycle.
dNTPs: Usually equimolar concentrations (200-250 µM each) of dATP, dCTP,
dGTP and dTTP are used. Taq adds these dNTPs to the growing DNA strands
according to the complementarity of the template DNA strand.
Template DNA: 50-100 ng of pure DNA is usually used as the starting material for any PCR reaction. The
DNA should be free of any contaminating proteins which can inhibit the reaction.
PCR machines or thermal cyclers are used to vary the temperatures cyclically thus helping in
automation of the process. After ‘n’ number of cycles, theoretically, 2n number of copies of the target
DNA will be formed. However the efficiency of the process almost never reaches 100 %

PCR – STEPS AND PRINCIPLE

Denaturation step: Double-stranded DNA templates denature at a temperature that is determined in

part by their G+C content. The higher the proportion of G+C, the higher the temperature required to
separate the strands of template DNA. In PCRs catalyzed by Taq DNA polymerase, denaturation is
carried out at 94-95°C, which is the highest temperature that the enzyme can endure for 30 or more
cycles without sustaining excessive damage. It causes melting of DNA template and primers by
disrupting the hydrogen bonds between complementary bases of the DNA strands, yielding single
strands of DNA.

Annealing step: After the initial denaturation, the temperature is rapidly brought
down to the annealing temperature when there is annealing of the template DNA and the primers.
Annealing is usually carried out 3-5OC lower than the calculated melting temperature at which the
oligonucleotide primers dissociate from their templates. Stable DNA-DNA hydrogen bonds are only
formed when the primer sequence very closely matches the template sequence. The polymerase binds
to the primer-template hybrid and begins DNA synthesis. The temperature used for the annealing step is
critical. If the annealing temperature is too high, the oligonucleotide primers anneal poorly, if at all, to
the template and the yield of amplified DNA is very low. If the annealing temperature is too low,
nonspecific annealing of primers may occur, resulting in the amplification of unwanted segments of DNA

Extension/elongation step: At this step the DNA polymerase synthesizes a new DNA strand
complementary to the DNA template strand by adding dNTPs that are complementary to the template
in 5' to 3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the
end of the nascent (extending) DNA strand. The extension time depends on the length of the DNA
fragment to be amplified. Extension is carried out at or near the optimal temperature for DNA synthesis
catalyzed by the thermostable polymerase, which in the case of Taq DNA polymerase is 72-78°C. In the
first two cycles, extension from one primer proceeds beyond the sequence complementary to the
binding site of the other primer. In the next cycle, the first molecules are produced whose
length is equal to the segment of DNA delimited by the binding sites of the primers. From the third cycle
onward, this segment of DNA is amplified geometrically. The polymerization rate of Taq polymerase is
approximately 2000
nucleotides/minute at the optimal temperature.

TYPES OF PCR

1. REVERSE TRANSCRIPTASE PCR (RT-PCR)

2. REAL TIME PCR
3. Nested polymerase chain reaction (PCR)
4. Multiplex PCR

REVERSE TRANSCRIPTASE PCR : RT-PCR is a method used to amplify cDNA copies of RNA. An
oligodeoxynucleotide primer is hybridized to the mRNA and is then extended by an RNA-dependent DNA
polymerase to create a cDNA copy that can be amplified by PCR. Depending on the purpose of the
experiment, the primer for first-strand cDNA synthesis can be specifically designed to hybridize to a
particular target gene or it can bind generally to all mRNAs (examples include oligodT primers which
bind to the polyA tails of mRNAs only and random hexamers which bind randomly to all the RNA
molecules present). The enzyme used in RT-PCR is usually MMLV RT (Moloney strain of murine leukemia
virus reverse transcriptase) which has an optimum temperature of activity at 37°C – 42°C. RT-PCR is used
to clone the 5' and 3' termini of mRNAs and to generate large cDNA libraries from very small amounts of
mRNA. RT-PCR can also be adapted to identify mutations in transcribed sequences and to measure the
gene expression levels

Real time PCR: Real time PCR (also called kinetic PCR) is generally used to quantify gene expression. It is
also used to screen for mutations and single nucleotide polymorphisms. Real time PCR uses
fluorescence-detecting thermocyclers to amplify specific nucleic acid sequences and measure their
concentration simultaneously. The instrument plots the rate of accumulation of amplified DNA over the
course of an entire PCR. The greater the initial concentration of target sequences in the reaction
mixture, the fewer the number of cycles required to achieve a particular yield of amplified product. The
initial concentration of target sequences can therefore be expressed as the fractional cycle number CT
required to achieve the present threshold of amplification. A plot of CT, against the log of the initial copy
number of a set of standard DNAs yields a straight line. The target sequences in an unknown sample
may be easily quantified by interpolation into this standard curve. The ability to quantify the amplified
DNA during the exponential phase of the PCR, when none of the components of the reaction are
limiting, results in an improved precision in the quantitation of target sequences.