PCR explained in simple terms - A T G & C of PCR - Question and answers PCR

PCR in simple terms
The AT G & C of PCR
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What is PCR?
• PCR is polymerase Chain Reaction.

Which Polymerase used in PCR?
• There are various types of polymerases used in PCR, most commonly used
polymerase is the enzyme fromThremus aquaticus –Taq Polymerase.
• Other polymerases used includes – BST Polymerase, pfu polymerase, etc.

WhyTaq Polymerase why not any other
polymerases?
• Taq Polymerase is a thermostable enzyme, can work well even at higher
temperatures, most of the enzymes denature at higher temperatures, that’s
why taq polymerase is used in PCR.

Why is it called chain reaction?
• In PCR, DNA doubles after each cycle, leading to exponential increase in the
total no of DNA copies.That’s why it is called chain reaction.
• Amplification is expressed by general equation 2^n; where n is the no of
cycles

How does PCR work?
• PCR works by these 3 steps:
• 1. Denaturation
• 2. Annealing
• 3. Extension

What is DNA Denaturation?
• Denaturation is the separation of double stranded DNA to two single
strands.
• DNA can be denatured by heating into high temperatures (~95°C), resulting
is the breakage of hydrogen bonds and separation of the double strand into
single strand.

Structure of double stranded DNA (dsDNA)

What are primers?
• Primers are short stretch of oligonucleotides.
• Primers are the starting point to which polymerase starts adding nucleotides.
• Primers are selected from the target sequence to be amplified, it is selected in such
way that the primers will bind only to the specific target.
• Two primers are used in PCR, Forward Primer and Reverse Primer.

What is annealing?
• Annealing is process of primer binding to DNA.

Annealing

What is Extension?
• Once the primer binds to the target DNA, polymerase starts adding
nucleotides to the primer causing the strand to extend.

Extension

What are the reagents used in PCR reaction?
• Buffer
• dNTPs (deoxy nucleotide triphosphates)
• Taq Polymerase
• Primers

PCR simple maths
• PCR DNA doubling: 2^n;
• Calculate the no of DNA molecules produced after 30cycles of PCR:
• Starting copy = 1;
• No of copies generated after n cycles = (starting no. of copies) * (2^n)
= 1*2^30
= 1073741824 copies; or 1.07*10^9 copies

How do I know PCR has worked?
Multiple Options:
• Perform agarose gel electrophoresis after PCR.
• Use Intercalating dye in the PCR reaction and monitor fluorescence.
• Use Oligonucleotide Probes – Fluorescent Probes / Non Fluorescent Probes.

What is real time PCR?
• Real time PCR also known as quanititavie PCR or qPCR is a type of PCR in
which the DNA amplification is monitored real time using intercalating dyes
or oligonucleotide fluorescent probes.

Amplification Profile – real time PCR

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