Polymerase Chain Reaction (PCR)

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  • Abstract: Hello and welcome friends to my article. Today will discuss a very important topic for many competitive exams like CSIR,GATE,NEET etc in life sciences stream. I will try to make you understand the matter in an easy way....

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Polymerase Chain Reaction


Polymerase chain reaction is a technique, used in molecular biology to amplify a single copy or few copies of particular DNA sequence. When there is a small amount of DNA then we use the method or technique for further analysis the DNA. Which can generate thousand to millions copies of particular DNA sequence. A basic PCR setup required several components and reagents.

when we use the technique in the lab then we first prepare a mixture of a reaction ......

The picture is downloaded from Shutterstock.
 

1. At the first we need a DNA template for amplification.

2. Primers ( primers are short oligonucleotide sequence which are 15 to 20 nucleotide long ) two DNA primers that are complementary to the three prime end of each of the strand of the DNA target. Specific primers that are complementary to the DNA target region selected beforehand.
Sometimes purchased from market( commercial biochemical supplier)

3. DNA polymerase. (It should be heat resistant) use taq polymerase or other polymerase.

4.dNTPs ( deoxyribonucleoside triphosphate, new types containing tri phosphate group ) the building blocks from which the DNA polymerase synthesized a new strand .
 
 
5. Buffer at optimum pH.
A buffer for the optimum activity with a suitable chemical environment.

Procedure : After preparing the reaction mixture with the PCR tube we perform a programming.

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Keep the picture on your mind .....

Let's discuss the process here in three steps 

Step 1. Denaturation - to take the DNA template and heat it at 90 to 95 degree Celsius temperature for the denaturation.
Denatured means breakdown of hydrogen bonds. 
 
Step 2. Annealing - Annealing of primers binds to 3 prime end of the template DNA at usually 50 to 70 degree Celsius . If the primer is more than 20 nucleotides then the binding affinity of the primer will be slow.

Step 3. Elongation - DNA polymerase synthesis in new DNA strand which is complementary to the DNA template adding with dntps from reaction mixture. The complementary strength of the template is 5 prime to 3 prime directions.

In this case of annealing , we have to follow some condition 
1. Length will be optimum

2.binding temperature of primers should be 5 degree less than the melting temperature of DNA.

3. We must have a known DNA melting temperature.
There is a formula to calculate the melting temperature that I put here 👇

Tm( melting temperature of DNA) = {4+(G+C) + 2×(A+T)} °C 
KNOWN SEQUENCE OF DNA IS NEEDED FOR THIS EQUATION.

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HOPE YOU PEOPLE UNDERSTAND THE PROCESS OF WHICH I HERE EXPLAIN. 💖💓

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