Polymerase Chain Reaction PCR- Principle, Steps, Requirements and Applications With MCQ II Helpful for various Biology Exams II LetsBloom study material

WHAT IS PCR ?

PCR stands for Polymerase Chain Reaction, is a method by which single strand of DNA is Multiplied or Amplified to provide a large Homogeneous DNA Sample.

By using a small specific DNA fragment a large amount of specific sequence of DNA is synthesized.

It is a method for In Vitro DNA Synthesis (in controlled environment outside organism) used for amplification of DNA.

The enzyme used for repeated cycles of duplication of DNA is DNA Polymerase, that's normally present in every organism.

Cell division is a natural form of PCR.

So, PCR is a technique that amplify exponentially a fragment of DNA via an enzymatic replication without any help of living organism.

A single piece of DNA can generate million to trillion DNA

REQUIREMENTS:

STEPS:

Denaturation: By Heating. This is the first step of PCR where double stranded DNA is heated at upto 94°C so that it gets separated into two strands. 

Primer Annealing: By Primers and require temperature ~67°C

Extension or DNA Synthesis: By Polymerase and require temperature ~72°C

The result is an exponential increase in amount of DNA fragment marked by 5' ends of primer

Since all the three steps takes place at different temperature so the whole requirements are placed in a Thermal Cycler that maintains the temperature as required in each step.

About DNA Polymerase:

The DNA Polymerase used is Thermostable and obtained from bacterium Thermophilus aquaticus that is a heat tolerant bacterium called as Taq Polymerase.

Taq Polymerase lacks 3' - 5' proofreading activity so it can make some mistakes.

Polymerase binds to a single strand of DNA.

Another type of polymerase that can be used is Pfu or Pwo Polymerase from Archaebacterium, and it has Proofreading activity.

So, Taq polymerase has speed but lacks proofreading activity while  Pwo Polymerase has lesser rate and high proofreading activity.

WATCH OUT COMPLETE EXPLANATION VIDEO FOR MORE DETAILS:

QUESTION PRACTICE

Which of the following correctly explains sequence of processes in PCR?

Primer annealing DNA Synthesis Denaturation

Primer annealing Denaturation DNA Synthesis

Denaturation Primer annealing DNA Synthesis

Denaturation DNA Synthesis Primer annealing

Polymerase Chain Reaction is usually used for? (RPSC)

Hybridization

Gene transfer

Amplification

DNA Fingerprinting

Which of the following cofactor is present in Taq Polymerase

Mg2+

Ca

EDTA

Cl

During PCR the amplification of Gene of Interest is catalyzed by enzyme (KVS)

Primase

Taq Polymerase

DNA Ligase

DNA dependent RNA Polymerase

Which of the following is most critical temperature, to be decided by primer sequence, in any PCR experiment?  ICAR PG Plant biotechnology 2022

Annealing temperature

Denaturation temperature

Primer extension temperature 

Final holding temperature