Molecular Biology is the field of biology that studies the composition, structure and interactions of cellular molecules – such as nucleic acids and proteins – that carry out the biological processes. Polymerase chain reaction (abbreviated PCR) is a laboratory technique for rapidly producing (amplifying) millions to billions of copies of a specific segment of DNA, which can then be studied in greater detail.

1. Sequencing andAmplification
of DNA
Chapter-4

2. This table contains the
difference between prokaryotic
and eukaryotic cells at different
characteristic levels. Archaea
bacteria are those which are
ancient and are known to
evolve from bacteria and blue-
green algae.

3. Difference between DNAand RNA

4. Polymerase Chain Reaction
(PCR)

5. What is PCR?
• It is an in vitro molecular biology technique to amplify
the target DNA sequence.
• The final product of the PCR is known as amplicon,
where the number of cycles can be varied to get the
desired copy number (i.e. 2^n).
• Three steps include: denaturation, annealing and
extension or polymerization.

6. PCR Components
• The important components of PCR includes:
1) Template DNA sequence- this includes any target sequence
single copy or multiple copies
2) DNA polymerase- Taq polymerase is the DNA polymerase
enzyme used as they can withstand high temperatures
3) Primers- short length of nucleotide sequences which has free
3’-OH end for adding deoxynucleotides
4) Deoxynucleotides- these are monomer units which is used/
added to the primers while extension
5) Mg2+ which acts as cofactor for DNA polymerase

8. Amplification of DNA
• Three steps are involved in polymerase chain reaction. They
are:
1. Denaturation- at 94ºC, where the double stranded DNA is
split into single strands which run in opposite direction i.e. 3’
to 5’ and 5’ to 3’
2. Annealing- takes place between 50ºC- 60ºC where primer
gets attached to the template strand
3. Extension- at 74ºC where the deoxynucleotides are added to
the primers

9. Types of PCR
• The different types of PCR are:
1. RT-PCR
2. Real time PCR
3. Quantitative real time PCR
4. Multiplex PCR
5. Nested PCR

10. Applications of PCR
• PCR can be used for genetic fingerprinting or DNA profiling
purpose in forensics.
• It is also useful in the diagnostics, where the pathogen
presence is identified.
• Less common organisms like viruses can be detected.

11. Thank You