Brief introduction to PCR (The Polymerase Chain Reaction) - Amplifying DNA

Polymerase chain reaction, or PCR, is a technique to make many copies (millions or billions) of a specific DNA
region in vitro (in a test tube rather than an organism).
The polymerase chain reaction (PCR) was originally developed in 1983 by the American biochemist Kary Mullis. He
was awarded the Nobel Prize in Chemistry in 1993 for his pioneering work. PCR is used in molecular biology to make
many copies of (amplify) small sections of DNA or a gene. Using PCR, it is possible to generate thousands to
millions of copies of a particular section of DNA from a very small amount of DNA.
PCR is a common tool used in medical and biological research labs. It is used in the early stages of processing DNA
for sequencing, for detecting the presence or absence of a gene to help identify pathogens during infection, and when
generating forensic DNA profiles from tiny samples of DNA.
Working principles behind every PCR: Five core ‘ingredients’ are required to set up a PCR.
(1) DNA template: the DNA template to be copied
(2) PCR primers: primers, short stretches of DNA that initiate the PCR reaction, designed to bind to either side of
the section of DNA you want to copy. PCR primers are short pieces of single-stranded DNA, usually
around 20 nucleotides in length. Two primers are used in each PCR reaction, and they are designed so that they flank
the target region (region that should be copied). That is, they are given sequences that will make them bind to opposite
strands of the template DNA, just at the edges of the region to be copied. The primers bind to the template by
complementary base pairing.

(3) DNA nucleotide bases (also known as dNTPs): DNA bases (A, C, G and T) are the building blocks of DNA and
are needed to construct the new strand of DNA
(4) Taq polymerase enzyme: PCR requires a DNA polymerase enzyme that makes new strands of DNA (to add in the
new DNA bases), using existing strands as templates. The DNA polymerase typically used in PCR is
called Taq polymerase, this enzyme is isolated from a heat-tolerant bacterium (Thermus aquaticus).
T. aquaticus lives in hot springs and hydrothermal vents. Its DNA polymerase is very heat-stable and is most active
around 70 °C (a temperature at which a human or E. coli DNA polymerase would be nonfunctional). This heat
stability makes Taq polymerase ideal for PCR. As we'll see, high temperature is used repeatedly in PCR
to denature the template DNA, or separate its strands.
(5) Buffer: to ensure the right conditions for the reaction.
The steps of PCR: The key ingredients of a PCR reaction are Taq polymerase, primers, template DNA, and
nucleotides (DNA building blocks). The ingredients are assembled in a tube, along with cofactors needed by the
enzyme, and are put through repeated cycles of heating and cooling that allow DNA to be synthesized. The basic steps
are:
Denaturation (96 °C): Heat the reaction strongly to separate, or denature, the DNA strands. This provides single-
stranded template for the next step.
• During this stage the cocktail containing the template DNA and all the other core ingredients is heated to 96⁰C.
• The high temperature causes the hydrogen bonds? between the bases in two strands of template DNA to break
and the two strands to separate.
• This results in two single strands of DNA, which will act as templates for the production of the new strands of
DNA.
• It is important that the temperature is maintained at this stage for long enough to ensure that the DNA strands
have separated completely.
• This usually takes between 15-30 seconds.
Annealing (55 - 65°C): Cool the reaction so the primers can bind to their complementary sequences on the single-
stranded template DNA.
• During this stage the reaction is cooled to 50-65⁰C. This enables the primers to attach to a specific location on the
single-stranded template DNA by way of hydrogen bonding (the exact temperature depends on the melting
temperature of the primers you are using).
• Primers are single strands of DNA or RNA? sequence that are around 20 to 30 bases in length.
• The primers are designed to be complementary? in sequence to short sections of DNA on each end of the
sequence to be copied.
• Primers serve as the starting point for DNA synthesis. The polymerase enzyme can only add DNA bases to a
double strand of DNA. Only once the primer has bound can the polymerase enzyme attach and start making the
new complementary strand of DNA from the loose DNA bases.
• The two separated strands of DNA are complementary and run in opposite directions (from one end - the 5’ end –
to the other - the 3’ end); as a result, there are two primers – a forward primer and a reverse primer.
• This step usually takes about 10-30 seconds.
Extension (72 °C): Raise the reaction temperatures so Taq polymerase extends the primers, synthesizing new strands
of DNA.
• During this final step, the heat is increased to 72⁰C to enable the new DNA to be made by a special Taq DNA
polymerase enzyme which adds DNA bases.
• Taq DNA polymerase is an enzyme taken from the heat-loving bacteria? Thermus aquaticus.
✓ This bacterium normally lives in hot springs so can tolerate temperatures above 80⁰C.
✓ The bacteria's DNA polymerase is very stable at high temperatures, which means it can withstand the
temperatures needed to break the strands of DNA apart in the denaturing stage of PCR.
✓ DNA polymerase from most other organisms would not be able to withstand these high temperatures, for
example, human polymerase works ideally at 37˚C (body temperature).
• 72⁰C is the optimum temperature for the Taq polymerase to build the complementary strand. It attaches to the
primer and then adds DNA bases to the single strand one-by-one in the 5’ to 3’ direction.
• The result is a brand-new strand of DNA and a double-stranded molecule of DNA.
• The duration of this step depends on the length of DNA sequence being amplified but usually takes around one
minute to copy 1,000 DNA bases (1Kb).
• These three processes of thermal cycling are repeated 20-40 times to produce lots of copies of the DNA sequence
of interest.
• The new fragments of DNA that are made during PCR also serve as templates to which the DNA polymerase
enzyme can attach and start making DNA.
• The result is a huge number of copies of the specific DNA segment produced in a relatively short period of time.
This cycle repeats 25 - 35 times in a typical PCR reaction, which generally takes 2 - 4 hours, depending on the length
of the DNA region being copied. If the reaction is efficient (works well), the target region can go from just one or a
few copies to billions.
That’s because it’s not just the original DNA that’s used as a template each time. Instead, the new DNA that’s made in
one round can serve as a template in the next round of DNA synthesis. There are many copies of the primers and many
molecules of Taq polymerase floating around in the reaction, so the number of DNA molecules can roughly double in
each round of cycling. This pattern of exponential growth is shown in the image below.
Results of PCR: The results of a PCR reaction are usually visualized (made visible) using gel electrophoresis. Gel
electrophoresis is a technique in which fragments of DNA are pulled through a gel matrix by an electric current, and
it separates DNA fragments according to size. A standard, or DNA ladder, is typically included so that the size of the
fragments in the PCR sample can be determined.
DNA fragments of the same length form a "band" on the gel, which can be seen by eye if the gel is stained with a
DNA-binding dye.
Applications of PCR: PCR is used in many research labs, and it also has practical applications in forensics, genetic
testing, and diagnostics. For instance, PCR is used to amplify genes associated with genetic disorders from the DNA
of patients (or from fetal DNA, in the case of prenatal testing). PCR can also be used to test for a bacterium or DNA
virus in a patient's body: if the pathogen is present, it may be possible to amplify regions of its DNA from a blood or
tissue sample.