Conventional PCR Virtual Lab

Theoretical Background / Context

•  Conventional Polymerase Chain Reaction is a technique for in vitro amplification of a specific short-defined segment of DNA. 
• Most PCR methods typically amplify DNA fragments of up to ~10kb. 
• PCR in microbiology is used to detect, identify, and study microorganisms by amplifying specific DNA sequences.
• In a test tube, a pair of oligonucleotide primers directs the polymerase enzyme and the deoxyribonucleotides to a specific gene in the DNA sample. 
• A replication-like process occurs in cycles. This repetition results in DNA amplification and as many as a billion copies can be the result of 30 cycles. 
• Therefore, minimal amounts of DNA can be detected. 

Principle of Work

A PCR virtual lab set up requires several components and reagents.

These components include:

• Two primers (Forward and reverse).
• Taq polymerase.
• Deoxynucleoside triphosphates (dNTPs).
• Buffer solution.
• Distilled water.
• Template DNA.

In PCR virtual lab,the PCR reaction follows the following steps:

1. Denaturation: high temperature (95 degrees centigrade) allows the separation of the two DNA strands due to the breakdown of hydrogen bonds linking bases together.
2. Primer annealing:  primers are short, synthetic sequences of single-stranded DNA typically consisting of 20-30 bases.  Annealing usually takes place between 40 degrees Centigrade and 65 degrees centigrade, depending on the length and base sequence of the primers.

• Extension (elongation): Taq polymerase is a recombinant thermo-stable DNA polymerase, from the organism Thermus aquaticus. It is active at high temperatures. Temperature is raised at this stage to 72 degrees centigrade and complementary dNTPs are added starting at the 3’ end of the primer. Hence; Taq DNA polymerase was synthesized in the 5’-3’ direction, making this technique essential for PCR in microbiology applications.