Polymerase Chain Reaction ( PCR )

This is a method to produce very large numbers of copies of specific DNA sequences without cloning. Therefore PCR can amplify specific sequences or add sequences such as  endonuclease  recognition  sequences as primers to cloned DNA.

PCR consists of 5 main components.

-Target DNA

-Single stranded Oligonucleotides (primers)

-d NTPs (dATP, dCTP, dTTP, dGTP)

-Taq DNA polymerase

-Termocycler

There are three main steps in PCR.

Step1:  Denaturation. The mixture of excess primer and DNA fragment is heated to about 95° C causing the double strands of target DNA to be denatured into single strands.

Step 2: Annealing of Primers. The temperature is dropped down between about 35⁰- 65°C

As the temperature decreases the single strands of DNA reassociate into double strands. Large excess of primer allows two primers anneal or bind to their complementary sequences on the target DNA leaving the rest of the fragment single-stranded.

Step 3: Primer Extension. The temperature is raised to 70⁰- 75°C Taq  polymerase is added. Taq polymerase extends the primer into a complementary copy of the entire single-stranded fragment, in the 5’-3’ direction.  As both the DNA strands are replicated, two copies of the original fragment are gained.

This process is repeated many times. At each time, the number of DNA copies doubles. This is continued until enough copies are gained for the analysis.