How does PCR work?
- The principles behind every PCR, whatever the sample of DNA, are the same.
- Five core ‘ingredients’ are required to set up a PCR. We will explain exactly what each of these do as we go along. These are:
- the DNA template to be copied
- primers, short stretches of DNA that initiate the PCR reaction, designed to bind to either side of the section of DNA you want to copy
- DNA nucleotide bases (also known as dNTPs). DNA bases (A, C, G and T) are the building blocks of DNA and are needed to construct the new strand of DNA
- Taq polymerase enzyme to add in the new DNA bases
- buffer to ensure the right conditions for the reaction.
- PCR involves a process of heating and cooling called thermal cycling which is carried out by machine.
- There are three main stages:
- Denaturing – when the double-stranded template DNA is heated to separate it into two single strands.
- Annealing – when the temperature is lowered to enable the DNA primers to attach to the template DNA.
- Extending – when the temperature is raised and the new strand of DNA is made by the Taq polymerase enzyme.
- These three stages are repeated 20-40 times, doubling the number of DNA copies each time.
- A complete PCR reaction can be performed in a few hours, or even less than an hour with certain high-speed machines.
- After PCR has been completed, a method called electrophoresis can be used to check the quantity and size of the DNA fragments produced.
No comments:
Post a Comment