Daksh Jain B015
FST JDSOLA
Polymerase Chain Reaction: A Revolutionary Technique
Polymerase Chain Reaction (PCR), is a process invented by Kary B. Mullis in 1983,
which is considered to be one of the most important scientific progressions in molecular
biology. It is used to rapidly make millions to billions of copies of a specific DNA sample,
allowing scientists to take specific segments of DNA and amplify it to a large enough
amount to study it in detail. For the importance of this invention, Mullis was awarded the
Navel Prize for Chemistry in 1993. This process is relatively fast and inexpensive and
has a wide range of applications because studies of isolated pieces of DNA are nearly
impossible without PCR amplification.
PCR has become an indispensable technique used for a broad variety of applications:
1. Forensics: The DNA from body fluids, hair or tissue samples at the crime scene
is amplified to create a unique pattern known as DNA fingerprint, different for
each individual and is compared to the suspects.
2. Paternity testing: An individual's DNA is matched with their close relatives identify
relations and can be used to confirm the biological parents of an adopted child. It
can also be used to identify historical family relationships
3. Disease Diagnosis: PCR can be used for rapid and highly effective diagnosis of
infectious diseases, including those caused by bacteria or viruses. It can also be
used to test prospective parents for being genetic carriers
4. Disease treatment: PCR can be used to detect the amount of the pathogen
causing the disease to provide medication according to its severity.
5. Wildlife conservation: PCR can be used to monitor products made from
endangered species. It can be used to identify ecosystems for the presence of
certain species
To understand PCR, it is imperative to understand DNA. Deoxyribonucleic acid (DNA) is
a molecule that contains genetic instructions for the development, functioning, growth
and reproduction of all known organisms. DNA is passed from adult organisms to their
offspring during reproduction. DNA is found inside the nucleus of the cell, where it is
tightly packaged in the form of chromosomes. DNA is made up of nucleotides, which
contain a sugar called deoxyribose, a phosphate group and one of the four nitrogen-
containing nucleobases cytosine [C], guanine [G], adenine [A] or thymine [T]. The two
polynucleotide chains coil around each other to form a double helix. The nucleotides are
joined to one another in a chain by covalent bonds and the nitrogenous bases of the two
separate polynucleotide strands are bound together, according to base pairing rules (A
with T and C with G), with hydrogen bonds to make double-stranded DNA. A gene is a
sequence of these bases that contains biological information and can influence the
characteristics of an organism. The gene determines what biological instructions are
contained in a strand of DNA.
DNA's information is used to make proteins by a process known as transcription. It is
achieved through complementary base pairings. DNA strands are used to create
messenger RNA strands where DNA bases are exchanged for their complementary
bases, except in the case of thymine, where Uracil is substituted in mRNA. These
mRNA strands are used to determine the sequence of amino acids in proteins, in the
process known as translation.
This way, studying DNA can give crucial information about the organism and PCR helps
us achieve this. So, how does this technique work? It requires several ingredients:
1. The most obvious is the DNA template, the sample that contains the specific
DNA target region to amplify.
2. A DNA polymerase, an enzyme to polymerize the new DNA strands, which acts
as a catalyst in the process. Because the process relies on thermal cycling, Taq
polymerase is used as it can withstand high temperatures.
3. DNA primers, complementary to the ends of each of the strands of the DNA
target and not complementary to each other. These are single-stranded
sequences much shorter than the target region.
4. Deoxynucleoside triphosphates (dNTPs), the building blocks of the new DNA
strands.
5. A buffer solution to provide suitable environment for optimum action and stability
of the polymerase.
6. Magnesium ions (Mg2+)
All this material is collected in small tubes and placed in a thermal cycler. PCR consists
of a series of repeated temperature changes, known as thermal cycles, which have
three steps at different temperatures:
1. Denaturation: The Chamber is heated to 95°C which breaks the hydrogen bonds
between complementary bases, leading to the separation of the double-stranded
DNA into two single-stranded molecules.
2. Annealing: The temperature is lowered to 50°C-60°C to allow the primers to
attach to the ends of each of the single-stranded DNA. During this step, the Taq
polymerase binds to the primers and begin DNA formation.
3. Extension: The temperature is raised to 72°C allowing the polymerase to
synthesize a new DNA strand using the original strands a templates by adding
dNTPs as building blocks.
The result is the formation of one additional copy of DNA per template. These can be
used to create further copies. The cycle is repeated till the desired number of copies is
achieved.