DEPARTMENT OF MICROBIOLOGY

ST. XAVIER’S COLLEGE (AUTONOMOUS), KOLKATA

30, MOTHER TERESA SARANI, KOLKATA – 700016

NAME: SOUNAK DAS

ROLL NO.: 3-16-17-0629
REGN. NO.: A01-1112-0877-17
SUBJECT: MB36172T
MODULE: 17
SEMESTER: VI
 PCR AND ITS APPLICATION IN RECOMBINANT
DNA TECHNOLOGY

Introduction:
Polymerase Chain Reaction (PCR) is a technique by which any specific genetic
element can be amplified to enormous quantity which further helps in analysis.
PCR was invented by American biochemist Kary Banks Mullis in 1984. Since,
then it has been used extensively in Medical Diagnostics, Forensic analysis,
Recombinant DNA Technology and scientific laboratories.
In PCR a thermostable DNA Polymerase called Taq Polymerase is used to
amplify the sample DNA. Taq Polymerase obtained from thermophilic bacteria
Thermus aquaticus can maintain optimal activity around 75oC – 80oC. A typical
PCR involves the following components; -
➢ DNA Template containing the target DNA region
➢ Heat tolerant DNA Polymerase (Taq Polymerase)
➢ Primers
➢ Deoxynucleotide triphosphates (dNTPs)
➢ Bivalent cations like Mg2+ and Mn2+

Fig. Stages of Polymerase Chain Reaction (PCR) cycle

(Source: https://www.bosterbio.com/media/images/MB_PCR_components.png)
Basic steps involved in PCR are as follows; -
1. Denaturation:
The sample is heated at around 95oC to denature the template DNA. Single
stranded template is acquired which is essential for the next step.
2. Annealing:
The sample is now cooled around 550C – 65oC to facilitate bonding of
primer to complementary sequences.
3. Extension:
The temperature is raised around 72oC to increase the activity of Taq
Polymerase. The enzyme extends the primer by adding dNTPs
complementary to the template strand. As a result, target DNA fragment is
synthesised using this method.
Denaturation, Annealing and Extension represents a single cycle. Billions of
copies of the target DNA can be achieved by running the cycle just 30 times. In
special cases, some steps are modified in accordance with the need of
operator. The biochemical reaction progression is further divided into three
phases; -
a) Exponential phase:
In this stage all reactants are in abundance with fully functional DNA
Polymerase enzyme. Thus, we observe 100% efficiency with sample
doubling after each cycle.
b) Linear phase:
In this stage, the enzymes start to lose its activity and concentration of
primers and dNTPs are reduced. Thus, these now acts as the limiting factor
for the reaction.
c) Plateau phase:
In this phase, no new DNA is synthesised due to exhaustion of reactants and
depleted enzymatic activity.