Polymerase Chain

Reaction (PCR)
Background
• The Polymerase chain reaction (PCR), first envisaged in 1984 by Kary
Mullis, has revolutionized life sciences and has become an essential
technique in many aspects of science, including clinical diagnostics,
forensics and genetic engineering. Kary Mullis eventually received the
Nobel Prize in Chemistry in 1993.
• PCR allows scientist to make unlimited copies of DNA fragments and
genes from a single copy of initial DNA. Each cycle of the polymerase
chain reaction doubles the number of copies of the gene of interest,
so for this experiment, which has 33 cycles, over 17 billion copies of
your gene of interest will be made for each starting template.
Working principle of PCR
• As the name implies, it is a chain reaction, a small fragment of the DNA section
of interest needs to be identified which serves as the template for producing the
primers that initiate the reaction.
• One DNA molecule is used to produce two copies, then four, then eight and so
forth. This continuous doubling is accomplished by specific proteins known as
polymerases, enzymes that are able to string together individual DNA building
blocks to form long molecular strands.
• To do their job polymerases require a supply of DNA building blocks, i.e., the
nucleotides consisting of the four bases adenine (A), thymine (T), cytosine (C)
and guanine (G).
• They also need a small fragment of DNA, known as the primer, to which they
attach the building blocks as well as a longer DNA molecule to serve as a
template for constructing the new strand. If these three ingredients are supplied,
the enzymes will construct exact copies of the templates.
The exponential copying of a gene of interest during the polymerase chain reaction
Components of PCR
1. A thermostable DNA polymerase to catalyze template-dependent
synthesis of DNA:
PCR utilizes the natural function of polymerase enzymes. In a normal dividing
cell, the copying of the genes requires a series of enzyme mediated reactions:
1. The DNA strands are unwound (denatured) by enzymes to form two
single strands.
2. A RNA polymerase binds and synthesizes a short complementary piece of
RNA on the DNA strand at the initiation site of replication.
3. This DNA/RNA heteroduplex acts as a priming site for the DNA
polymerase that binds and produces the complementary strand.
Cont’d…
• The key to the polymerase chain reaction was first discovered in 1976.
• The key is the Taq polymerase that was purified from the thermophile
Thermus aquaticus.
• A thermophile is an organism that grows at extreme temperature
(>100°C).
• The importance of the Taq polymerase being purified from a
thermophile is that the enzyme will not be destroyed at high
temperatures required to denature the DNA and allow PCR to begin.
Cont’d…
2. A pair of synthetic oligonucleotides to prime DNA synthesis:
• Design of the oligonucleotide primer being the most important factor
that influence the efficiency and specificity of the amplification
reaction.
• Careful designing of primers is required to obtain the desired products
in high yield, to suppress amplification of unwanted sequences and to
facilitate subsequent manipulation of the amplified product.
Cont’d…
3. Deoxynucleoside triphosphates(dNTPs)
• Standard PCRs contain equimolar amounts of all four dNTPs (dATP, dCTP, dGTP and dTTP).
• Concentrations of 200-250μM of each dNTP recommended for Taq polymerase in
reactions containing 1.5 mM MgCl2.
• High concentrations of dNTPs (>4mM) are inhibitory, perhaps because of sequestering of
Mg2+. However, a satisfactory amount of amplified product can be produced with dNTP
concentrations as low as 20μM- 0.5-1.0pM of an amplified fragment ~1 kb in length.
• To avoid problems, stocks of dNTPs should be stored at -20oC in small aliquots that should
be discarded after the second cycle of freezing or thawing.
• During long term storage at -20oC, small amounts of water evaporate and then freeze on
the walls of the vial. To minimize changes in concentration, vials containing dNTP
solutions should be centrifuged, after thawing, for a few seconds in a micro centrifuge.
Cont’d…
4. Divalent cations
• All thermostable DNA polymerases require free divalent cations- usually Mg2+ for activity. Some
polymerases will also work, though less efficiently with buffers containing Mn2+.
• Calcium ions are quite ineffective. Because dNTPs and oligonucleotides bind Mg2+, the molar
concentration of the cation must exceed the molar concentration of phosphate groups contributed by
dNTPs and primers.
• It is therefore impossible to recommend a concentration of Mg2+ that is optional in all circumstances.
• Although a concentration of 1.5 mM of Mg2+ is routinely used, increasing the concentration of Mg2+
to 4.5 mM or 6mM has been reported to decrease nonspecific priming in some cases and to increase it
in others.
• The optimal concentration of Mg2+ must therefore must be determined empirically for each
combination of primers and template. The preparations of template DNA should not contain
significant amount of chelating agents (like EDTA or negatively charged ions like PO43-), which can
sequester Mg2+.
Cont’d…
5. Buffer 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 of 10mM. When incubated
at 72oC(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 50mM KCl and works well for amplification
of segments of DNA >500bp in length. Raising the KCl concentration to
~70-100mM often improves the yield of shorter DNA segments.
Cont’d…
7. Template DNA
• Template DNA containing target sequences can be added to PCR in single or double
stranded form.
• Closed circular DNA templates are amplified slightly less efficiently than linear DNAs.
• Although the size of the template DNA is not critical, amplification of sequences
embedded in high molecular weight DNA(>10kb) can be improved by digesting the
template with a restriction enzyme that doesn't cleave within the target sequence.
• When working at its best, PCR requires only a single copy of target sequence as
template. More typically, however, several thousand copies of the target DNA are seeded
into the reaction.
• In the case of mammalian genomic DNA, upto 1μg of DNA is utilized per reaction. The
typical amounts of yeast, bacterial and plasmid DNAs used per reaction are 10μg, 1μg
and 1pg respectively.
Steps of PCR
PCR consists of 3 steps.
1) denaturation of the template by heat,
2) annealing of the oligonucleotide primers to the single stranded
target sequence(s),
3) and extension of the annealed primers by a thermostable DNA
polymerase.
1. Denaturation

• 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.
• The longer the DNA molecules, the greater the time required at the chosen
denaturation temperature to separate the two strands completely.
• If the temperature for denaturation is too low or if the time is too short at
AT rich regions of the template DNA will be denatured.
• When the temperature is reduced later in the PCR cycle, the template DNA
will reanneal into fully native condition.
Cont’d…
• In PCRs catalyzed by Taq polymerase, denaturation is carried out at 94-95oC,
which is the highest temperature that the enzyme can endure for 30 or more
cycles without sustaining excessive damage.
• In the first cycle of PCR, denaturation is sometimes carried out for 5 minutes to
increase the probability that long molecules of template DNA are fully
denatured.
• However this extended period of denaturation temperature is unnecessary for
linear DNA molecules as it may be deleterious sometimes.
• Denaturation for 45 seconds at 94-95C is routinely used to amplify linear DNA
molecules whose GC content is <55% and higher temperature for template
and/or target DNAs whose GC content is >55%. So much more heat tolerant
polymerases are preferred in such cases.
2. Annealing
• The second step is hybridization or annealing.
• The Taq polymerase requires a short piece of RNA to initiate DNA replication,
which in a normal cell is synthesized by the RNA polymerase.
• In the PCR reaction, short complimentary double stranded oligos are added
that bind the denatured DNA and act as origins of replications.
• These double stranded oligos are known as primers and are complimentary to
sequences up and down stream of the gene of interest.
• Two primers are used, one for each strand of DNA. Following denaturation,
the reaction mixture is rapidly cooled to a temperature below the melting
point of the specific primers (~55°C), below this temperature the primers bind
to their complementary bases on the now single stranded DNA.
Cont’d…
• 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, non specific annealing of
primers may occur, resulting in the amplification of unwanted
segments of DNA.
3. Extension
• In the third step, the temperature of the reaction is raised to the optimal
temperature for the polymerase (68‐72°C).
• The polymerase synthesizes new DNA, starting from the primer, the
polymerase reads a template strand and generates complementary
nucleotides very quickly.
• The result is two new helixes in place of the first, each composed of one
of the original strands plus its newly assembled complementary strand.
• The polymerase chain reaction is able to produce large copies of the
genes of interest as the above cycle can be repeated numerous times
leading to an exponential increase in the number of new copies
Steps of PCR
Thermocycler ------ PCR Machine
• The thermocycler is the most important piece of technology for
researchers wanting to use PCR.
• A thermocycler tightly regulates the temperature changes required
for denaturation, annealing and extension. It also controls the
number of cycles.
• Today’s thermocyclers are fully programmable and allow for rapid
heating and cooling and therefore tighter control of the PCR.