Once a DNA fragment has been isolated or produced (see the last article), the next step is often to produce many copies of it. One common way to do this is to use the polymerase chain reaction, which is a fairly simple process that can be automated. In this article we will look in detail at what happens to the DNA during the reaction.
Polymerase chain reaction (PCR)
The polymerase chain reaction (PCR) is a method to amplify DNA fragments in vitro (outside of a living organism). It can very quickly turn one DNA fragment into millions, and it works a bit like DNA replication.
A new word for this topic is a primer. A primer is a short section of single-stranded DNA which is designed to complementary base pair to the beginning of the DNA fragment you want to amplify. Two primers (one for each strand of the fragment) are needed. A reaction mixture is prepared to contain the DNA fragment, free DNA nucleotides, the primers, and a heat-stable version of DNA polymerase.
The PCR process is as follows:
- In the PCR machine, the mixture is heated to 95°C to break the hydrogen bonds in the DNA fragment and separate the strands.
- The mixture is cooled to somewhere between 50°C and 65°C to allow the primers to anneal to the DNA strands (they bind with complementary base pairing). The exact temperature depends on the primers and sometimes needs some optimisation.
- The mixture is heated to 72°C which is the optimum temperature for this type of DNA polymerase. Complementary base pairing of the free DNA nucleotides to the template strands ensures they are in the right order, and DNA polymerase joins them together with phosphodiester bonds (remember this is a condensation reaction), using the primers as a starting point.
- The cycle is complete, and the next cycle begins from step 2. Each cycle doubles the amount of DNA, and typically about 30 to 40 cycles will be run.
To work out how many DNA strands you are left with after 35 cycles (assuming you started with two strands) you can calculate 235, which is a huge number! So you can see how good the polymerase chain reaction is for producing many copies of DNA.
Summary
- PCR requires a DNA fragment, free DNA nucleotides, primers and DNA polymerase.
- Heating to 95°C separates the strands, cooling to 50°C – 65°C allows the primers to anneal, heating to 72°C allows DNA polymerase to join the free DNA nucleotides.
- Each cycle doubles the amount of DNA.
