2. Polymers:
Any of a class of natural or synthetic substances composed of very large molecules, called
macromolecules, that are multiples of simpler chemical units called monomers.
Polymerase:
An enzyme that synthesizes long chains of polymers or nucleic acids. DNA polymerase and RNA
polymerase are used to assemble DNA and RNA molecules, respectively.
Polymerase Chain Reaction (PCR):
A method widely used to make millions to billions of copies of a specific DNA sample rapidly,
allowing scientists to amplify a very small sample of DNA sufficiently to enable detailed study.
PCR was invented in 1983 by American biochemist Kary Mullis at Cetus Corporation.
PCR is a very sensitive technique that allows rapid amplification of a specific segment of DNA.
PCR makes billions of copies of a specific DNA fragment or gene, which allows detection and
identification of gene sequences using visual techniques based on size and charge.
3. •PCR relies on a thermostable DNA polymerase, Taq polymerase, and requires
DNA primers designed specifically for the DNA region of interest.
•The DNA polymerase typically used in PCR is called Taq polymerase, after the heat-tolerant
bacterium from which it was isolated (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.
•High temperature is used repeatedly in PCR to denature the template DNA, or separate its
strands.
•Taq polymerase can only make DNA if it's given a primer, a short sequence of nucleotides
that provides a starting point for DNA synthesis.
Basic Principle
4. • 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).
• 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.
5. Components Of PCR
Components Of PCR constitutes the following:
1.DNA Template– The DNA of interest from the sample.
2.DNA Polymerase– Taq Polymerase is used. It is thermostable and does not
denature at very high temperatures.
3.Oligonucleotide Primers- These are the short stretches of single-stranded
DNA complementary to the 3’ ends of sense and anti-sense strands.
4.Deoxyribonucleotide triphosphate– These provide energy for polymerization
and are the building blocks for the synthesis of DNA. These are single units of
bases.
5.Buffer System– Magnesium and Potassium provide optimum conditions for
DNA denaturation and renaturation. It is also important for fidelity, polymerase
activity, and stability.
6. Types of PCR
Real-time PCR
In this type, the DNA amplification is detected in real-time with the help of a fluorescent
reporter. The signal strength of the fluorescent reporter is directly proportional to the number of
amplified DNA molecules.
Nested PCR
This was designed to improve sensitivity and specificity. They reduce the non-specific binding of
products due to the amplification of unexpected primer binding sites.
Multiplex PCR
This is used for the amplification of multiple targets in a single PCR experiment. It amplifies
many different DNA sequences simultaneously.
Quantitative PCR
It uses the DNA amplification linearity to detect, characterize and quantify a known sequence in
a sample.
Arbitrary Primed PCR
It is a DNA fingerprinting technique based on PCR. It uses primers the DNA sequence of which
is chosen arbitrarily.
7. 1.Denaturation [96 °C]: Heat the reaction strongly to separate, or denature, the
DNA strands. This provides single-stranded template for the next step.
2.Annealing [55 - 65°C]: Cool the reaction so the primers can bind to their
complementary sequences on the single-stranded template DNA.
3. Extension (72 °C): Raise the reaction temperatures so Taq polymerase extends
the primers, synthesizing new strands of DNA.
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.
Steps of PCR
8.
9. 4. Visualization:
The results of a PCR reaction are usually
visualized (made visible) using gel
electrophoresis (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.
10. Applications of PCR:
Making DNA probes, Studying gene expression, Detection of viral and
bacterial infections, Diagnosis of genetic conditions, Detection of trace
amounts of DNA from tissue found at crime scene, Detection of DNA
from fossilized dinosaur tissue.
a.Diagnosis: Bacterial and viral diseases (TB, Hepatitis C, HIV)
b.Medicolegal / Forensiccases: DNA amplification from hair,
saliva, semen and blood sample (DNA fingerprinting).
c.Diagnosis of genetic disorders: Sickle Cell Disease,
thalassemia, cystic fibrosis
d.Prenatal diagnosis of inherited disorders
11. e. Cancer detection:
i. To monitor abnormal cells present in treated patients.
ii.Identification of mutation in onco suppressor genes
f. Fossil studies: To study evolution by comparing the sequences
in the extinct and living organism
